Method for the synthesis of a bifunctional complex

ABSTRACT

Disclosed is a method for obtaining a bifunctional complex comprising a display molecule part and a coding part, wherein a nascent bifunctional complex comprising a chemical reaction site and a priming site for enzymatic addition of a tag is reacted at the chemical reaction site with one or more reactants, and provided with respective tag(s) identifying the reactant(s) at the priming site is using one or more enzymes.

This application is a Continuation of U.S. Ser. No. 13/455,223 filed 25Apr. 2012, which is a Continuation of U.S. Ser. No. 10/525,817, filed 15Sep. 2005 (issued U.S. Pat. No. 8,206,901), which is a National Stage ofPCT/DK2003/00739 filed 30 Oct. 2003, which claims the benefit of U.S.provisional application Ser. No. 60/422,167, filed 30 Oct. 2002, U.S.provisional application Ser. No. 60/434,425, filed 19 Dec. 2002, U.S.provisional application Ser. No. 60/486,199, filed 11 Jul. 2003, SerialNo. PA 2002 01652, filed 30 Oct. 2002, Serial No. PA 2002 01955 filed 19Dec. 2002, and Serial No. PA 2003 01604 filed 11 Jul. 2003 in Denmark,and which applications are hereby incorporated by reference in theirentirety. To the extent appropriate, a claim of priority is made to eachof the above disclosed applications. All patent and non-patentreferences cited in these patent applications, or in the presentapplication, are hereby incorporated by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method for obtaining a bifunctionalcomplex comprising display molecule part and a coding part. Theinvention also relates to a method for generation of a library ofbifunctional complexes, a method for identifying a display moleculehaving a preselected property.

BACKGROUND

Approaches have been developed that allow the synthetic encoding ofpolypeptides and other biochemical polymers. An example of this approachis disclosed in U.S. Pat. No. 5,723,598, which pertains to thegeneration of a library of bifunctional molecules. One part of thebifunctional complex is the polypeptide and the other part is anidentifier oligonucleotide comprising a sequence of nucleotides whichencodes and identifies the amino acids that have participated in theformation of the polypeptide. Following the generation of the library ofthe bifunctional molecules, a partitioning with respect to affinitytowards a target is conducted and the identifier oligonucleotide part ofthe bifunctional molecule is amplified by means of PCR. Eventually, thePCR amplicons are sequenced and decoded for identification of thepolypeptides that have affinity towards the target. The library ofbifunctional complexes is produced by a method commonly known assplit-and-mix. The method implies that a linker molecule is divided intospatial separate compartments and reacted with a specific amino acidprecursor at one terminus in each compartment and appended a nucleicacid tag which codes for this specific amino acid precursor at the otherterminus by an orthogonal chemical reaction. Subsequently, the contentof the various compartments are collected (mixed) and then again splitinto a number of compartments for a new round of alternating reactionwith amino acid precursor and nucleotide tag. The split-and-mix methodis continued until the desired length of polypeptide is reached.

This prior art method is constrained in its application because theremust be compatible chemistries between the two alternating synthesisprocedures for adding a chemical unit as compared to that for adding anucleotide or oligonucleotide sequence. According to the prior art, theproblem of synthesis compatibility is solved by the correct choice ofcompatible protecting groups as the alternating polymers aresynthesised, and by the correct choice of methods for deprotection ofone growing polymer selectively while the other growing polymer remainsblocked.

Halpin and Harbury have in WO 00/23458 suggested another approach,wherein the molecules formed are not only identified but also directedby the nucleic acid tag. The approach is also based on the split-and-mixstrategy to obtain combinatorial libraries using two or more syntheticsteps. A plurality of nucleic acid templates are used, each having atone end a chemical reactive site and dispersed throughout the stand aplurality of codon regions, each of said codon regions in turnspecifying different codons. The templates are separated byhybridisation of the codons to an immobilised probe and subsequentlyeach of the strands is reacted at the chemical reaction sites withspecific selected reagents. Subsequently, all the strands are pooled andsubjected to a second partitioning based on a second codon region. Thesplit-and-mix method is conducted an appropriate number of times toproduce a library of typically between 10³ and 10⁶ different compounds.The method has the disadvantage that a large number of nucleic acidtemplates must be provided. In the event a final library of 10⁶different compounds is desired, a total of 10⁶ nucleic acid templatesmust be synthesised. The synthesis is generally cumbersome and expensivebecause the nucleic acids templates must be of a considerable length tosecure a sufficient hybridisation between the codon region and theprobe.

In WO 02/074929 a method is disclosed for the synthesis of chemicalcompounds. The compounds are synthesised by initial contacting atransfer unit comprising an anti-codon and a reactive unit with atemplate having a reactive unit associated therewith under conditionsallowing for hybridisation of the anti-codon to the template andsubsequently reacting the reactive units. Also this method suffers fromthe disadvantage that a large number of nucleic acid templates initiallymust be provided.

The prior art methods using templates suffer from the disadvantage thatencoding is dependent upon the recognition between the anti-codon andthe template. The hybridisation between two oligonucleotides can occurin the event there is a sufficient complementarity between these.Occasionally, the hybridisation will occur even though a complete matchbetween the oligonucleotides is not present. The effect is, in the eventa plurality of transfer units are present then sometimes the codonsequence of the template does not correspond to the reactive unitactually reacted. This undesired effect is even more pronounced when theformation of library is intended because a plurality of templates andbuilding blocks are supposed to find each other in the reaction media.When the hybridisation step is not completely correct, molecules will begenerated that are encoded by the incorrect codons on the template. Thiswill have two major effects on the selection process performed on thelibrary. First, templates with a codon combination encoding for bindingligands will be lost in the selection process. Secondly, and may be moreimportant, templates with a codon combination encoding for non-bindingligands will be enriched.

In an aspect of the present invention it is an object to provide anon-template dependent method for obtaining an encoded molecule, saidmethod allowing for versatile chemistries to be applied in the formationof the encoded molecule, because the application of compatibleorthogonal protection groups in the alternating formation of the encodedmolecule and oligonucleotide tag can be avoided. The present inventionin a preferred aspect intends to improve on the error pronehybridisation method previous suggested in the codon recognitionprocess. Furthermore, it is an object of the invention to reducenon-specific reaction products formed. Thus, in an aspect of the presentinvention, the present method has an inherent proof-reading facilitysecuring that the phenotype is accurately encoded by the genotype.

In some embodiments the bifunctional complex comprises codons ofdifferent lengths.

In some embodiments, instead of using enzymatic addition of a tag, thetag is chemically connected to the priming site applying a guidingoligonucleotide complementing an end of the tag and a part of thebifunctional complex comprising the priming site, such that the endsabut each other.

SUMMARY OF THE INVENTION

The present invention relates to a method for obtaining a bifunctionalcomplex comprising a display molecule part and a coding part, wherein anascent bifunctional complex comprising a chemical reaction site and apriming site for enzymatic addition of a tag is reacted at the chemicalreaction site with one or more reactants, and provided with respectivetag(s) identifying the reactant(s) at the priming site using one or moreenzymes.

Enzymes are in general substrate specific, entailing that the enzymaticaddition of a tag to the priming site is not likely to interfere withthe display molecule being formed. Thus, the application of protectiongroups on the coding part as well as the nascent display molecule can beavoided for this reason. However, it may be desired for other reasons toprotect the growing display molecule. Enzymes are available having anactivity in aqueous and organic media. The vast majority of enzymes,however, have a higher activity in an aqueous media compared to anorganic media. Therefore, prior to or subsequent to the providing of thetag it may be desired to change the media in order to obtain applicableconditions for the reaction of the reactant at the chemical reactionsite.

Generally, the display molecule part is formed by more than a singleround of reaction between one or more reactants and the chemicalreaction site. In a certain aspect of the invention, the nascentbifunctional complex reacted with one or more reactants and providedwith respective tag(s) is reacted further one or more times with one ormore reactant(s) and is provided with respective identifying tag(s) toproduce a reaction product as one part of the bifunctional complex andan identifying part comprising tags which codes for the identity of thereactants which have participated in the formation of the reactionproduct.

In a certain aspect of the invention, a round or cycle of reactionimplies that a single reactant is reacted with the chemical reactionsite and that a respective tag identifying the reactant is provided atthe priming site for enzymatic addition. In another aspect of theinvention, a round of reaction implies that multiple reactants arereacted at the chemical reaction site and that tags identifying one ormore, but not necessarily all, reactants are provided at the primingsite for enzymatic addition. The reaction at the chemical reaction siteand the addition of tags may occur in any order, i.e. the reaction mayoccur subsequent to, simultaneously with, or previous to the tagaddition. The choice of order may among other things be dependent on theenzyme type, the reaction conditions, and the type of reactant.

The nascent bifunctional complex comprises a chemical reaction site anda priming site for enzymatic addition of a tag. Optionally, the nascentbifunctional complex also comprises a linking moiety, which connects thechemical reaction site with the priming site. The linking moiety mayserve various purposes, such as distancing the priming site from thechemical reaction site sufficient from each other to allow an enzyme toperform the tag addition and provide for a hybridisation region. In anaspect of the invention, the linking moiety is a nucleic acid sequence.The length of the oligonucleotide is preferably suitable forhybridisation with a complementing oligonucleotide, i.e. the number ofnucleotides in the linking moiety is suitably 8 or above. In a certainembodiment, the linking moiety is attached to the chemical reaction sitevia a spacer comprising a selectively cleavable linker to enable adetachment of the display molecule from the coding part in a stepsubsequent to the formation of the final bifunctional complex. A nascentbifunctional complex is also referred to as a growing complex andspecifies an initial or intermediate complex to be processed accordingto the method of the present invention. An intermediate complexdesignates an initial complex that has been subjected to one or morerounds of reactant reaction and tag addition.

The chemical reaction site may comprise a single or multiple reactivegroups capable of reacting with one or more reactants. In a certainaspect the chemical reaction site comprises a scaffold having one ormore reactive groups attached. Examples of suitable reactive groupsinclude amine, carboxylic acid, thio, aldehyde, and hydroxyl groups.Examples of scaffolds include benzodiazepines, steroids, hydantiones,piperasines, diketopiperasines, morpholines, tropanes, cumarines,qinolines, indoles, furans, pyrroles, oxazoles, amino acid precursors,and thiazoles. Furthermore, the reactive groups of the chemical reactionsite may be in a pro-form that has to be activated before a reactionwith the reactant can take place. As an example, the reactive groups canbe protected with a suitable group, which needs to be removed before areaction with the reactant can proceed. A display molecule in thepresent description with claims indicates a chemical reaction site thathas been reacted with one or more reactants.

The reactants of the present invention include free reactants as well asreactants which comprises a functional entity and a nucleic acidsequence. The free reactant participates in the reaction with thechemical reaction site and may give rise to a chemical structure of thefinal display molecule. A functional entity attached to a nucleic acidmay be referred to herein as a building block and specifies a chemicalentity in which the functional entity is capable of being reacted at thechemical reaction site. In a certain aspect of the invention, thefunctional entity is detached from the nucleic acid part and transferredto the chemical reaction site. The oligonucleotide of the building blockmay or may not hold information as to the identity of the functionalentity. In a certain embodiment of the present invention, the reactantis a building block comprising an oligonucleotide sufficientcomplementary to the linking moiety to allow for hybridisation, atransferable functional entity, and an anti-codon identifying thefunctional entity. The free reactant is generally not attached to anucleic acid unless a nucleic acid component is intended in the finaldisplay molecule. The free reactant may have any chemical structure andpreferably comprises a reactive group or a precursor therefore, whichwill enable a reaction with a chemical reaction site. Examples ofreactive groups include hydroxyl groups, carboxylic acid groups, thiols,isocyanates, amines, esters, and thioesters. Optionally, a furtherreactant occurs to mediate a connection between the free reactant andthe chemical reaction site. The functional entity of a building blockresembles the free reactant as far as the requirement for reaction withthe chemical reaction site concerns. In addition, however, it is in mostinstances necessary to cleave the connection between the functionalentity and the nucleic acid following the reaction. Optionally, thereaction and cleavage may occur in a single step. Various types ofbuilding blocks are disclosed in detail below. In a certain aspect ofthe invention, the free reactant or the functional entity do not includea nucleotide.

The coding part of the nascent bifunctional complex is formed byaddition of at least one tag to a priming site using one or moreenzymes. Further tags may be attached to a previous tag so as to producea linear or branched identifier. As long as at least one tag of theidentifier is attached by an enzymatic catalysed reaction, further tagsmay be provided using chemical means or enzymatic means at thediscretion of the experimenter. In a certain embodiment of theinvention, all tags are provided using an enzymatic catalysed reaction.A tag suitably comprises recognition units, i.e. units which may berecognized by recognition groups. The recognition unit possess anability to carry information so as to identify a reactant. A variety ofdifferent kinds of recognition exist in nature. Examples are antibodies,which recognise an epitope, proteins which recognise another protein,mRNA which recognise a protein, and oligonucleotides which recognisecomplementing oligonucleotide sequences. Generally, it is preferred thatthe tag is a sequence of nucleotides.

The coding part of the bifunctional complex is in a preferred aspect ofthe invention amplifiable. The capability of being amplified allows forthe use of a low amount of bifunctional complex during a selectionprocess. In the event, the tag is a protein, the protein may beamplified by attaching the mRNA which has encoded the synthesis thereof,generating the cDNA from the mRNA and subjecting said mRNA to atranslation system. Such system is disclosed in WO 98/31700, the contentof which is incorporated herein by reference. An alternative method foramplifying a protein tag is to use phage displayed proteins. In general,however, the tag is a sequence of nucleotides, which may be amplifiedusing standard techniques like PCR. When two or more tags are present ina linear identifying oligonucleotide, said oligonucleotide generallyconsist of a certain kind of backbone structure, so as to allow anenzyme to recognise the oligonucleotide as substrate. As an example theback bone structure may be DNA or RNA.

The priming site of a nascent bifunctional complex is capable ofreceiving a tag. The chemical identity of the priming site depends amongother things on the type of tag and the particular enzyme used. In theevent the tag is a polynucleotide, the priming site generally comprisesa 3′-OH or 5′-phosphate group of a receiving nucleotide, or functionalderivatives of such groups. Enzymes which may be used for enzymaticaddition of a tag to the priming site include an enzyme selected frompolymerase, ligase, and recombinase, and a combination of these enzymes.

The reaction between the chemical reaction site and the one or morereactants may take place under suitable conditions that favours thereaction. In some aspects of the invention, the reaction is conductedunder hybridisation conditions, i.e. an annealing between twocomplementing oligonucleotides remains during the reaction conditions.In other aspects of the invention, the reaction is conducted underdenaturing conditions to allow for suitable condition for the reactionto occur. In the event, the coding part of the growing complex comprisesan oligonucleotide; said oligonucleotide is in an aspect of theinvention in a double stranded form during the reaction to reduce thelikelihood of side reactions between components of the oligonucleotideand reactants.

The tag identifying a reactant can be added to the priming site usingany appropriate enzyme. In a certain embodiment, a tag is provided atthe priming site of the nascent bifunctional complex utilizing anenzymatic extension reaction. The extension reaction may be performed bya polymerase or a ligase or a combination thereof. The extension using apolymerase is suitably conducted using an anti-tag oligonucleotide astemplate. The anti-tag oligonucleotide is annealed at the 3′ end of theoligonucleotide part of the nascent bifunctional complex with a singlestranded overhang comprising an anti-codon, which identifies thereactant. The anti-codon of the anti-tag can be transcribed to theidentifier part using a polymerase and a mixture of dNTPs.Alternatively, a ligase is used for the addition of the tag using one ormore oligonucleotides as substrates. The ligation can be performed in asingle stranded or a double stranded state depending on the enzyme used.In general it is preferred to ligate in a double stranded state, i.e.oligonucleotides to be ligated together are kept together by acomplementing oligonucleotide, which complements the ends of the twooligonucleotides.

Examples of suitable enzymes include DNA polymerase, RNA polymerase,Reverse Transcriptase, DNA ligase, RNA ligase, Taq DNA polymerase, Pfupolymerase, Vent polymerase, HIV-1 Reverse Transcriptase, Klenowfragment, or any other enzyme that will catalyze the incorporation ofcomplementing elements such as mono-, di- or polynucleotides. Othertypes of polymerases that allow mismatch extension could also be used,such for example DNA polymerase η (Washington et al., (2001) JBC 276:2263-2266), DNA polymerase τ (Vaisman et al., (2001) JBC 276:30615-30622), or any other enzyme that allow extension of mismatchedannealed base pairs. In another aspect, when ligases are used, suitableexamples include Taq DNA ligase, T4 DNA ligase, T4 RNA ligase, T7 DNAligase, and E. coli DNA ligase. The choice of the ligase depends to acertain degree on the design of the ends to be joined together. Thus, ifthe ends are blunt, T4 RNA ligase may be preferred, while a Taq DNAligase may be preferred for a sticky end ligation, i.e. a ligation inwhich an overhang on each end is a complement to each other.

The tag added to the priming site of the nascent bifunctional complexholds information as to the reactant. In the present invention withclaims, the information relating to the reactant will be termed codon.Apart from a combination of the nucleotides coding for the identity ofthe reactant, a tag may comprise further nucleotides. In a certainaspect of the invention, a tag comprises a framing sequence. The framingsequence may serve various purposes, such as an annealing region foranti-tags and/or as a sequence informative of the point in time of thesynthesis history the associated reactant has reacted.

The association between the codon and the identity of the reactant mayvary dependent on the desired output. In a certain embodiment, the codonis used to code for several different reactants. In a subsequentidentification step, the structure of the display molecule can bededuced taking advantage of the knowledge of the different attachmentchemistries, steric hindrance, deprotection of orthogonal protectiongroups, etc. In another embodiment, the same codon is used for a groupof reactants having a common property, such as a lipophilic nature,molecular weight, a certain attachment chemistry, etc. In a preferredembodiment however, the codon is unique, i.e. a similar combination ofnucleotides does not identify another reactant. In a practical approach,for a specific reactant, only a single combination of nucleotides isused. In some aspects of the invention, it may be advantageous to useseveral different codons for the same reactant. The two or more codonsidentifying the same reactant may carry further information related todifferent reaction conditions. In another aspect of the invention, asingle codon specifies two or more reactants.

In one aspect of the invention, each bifunctional complex is prepared bysimultaneous or sequentially tagging and reaction of reactant asillustrated in the scheme below:

x-X→ax-XA→1ax-XA1

Capital letters represent reactant or chemical reaction site. Lower caseletters represent tags.

A scaffold “X” is linked to a tag “x”. A reactant is linked to “X” e.g.“A” and so is a tag for that fragment e.g. “a”. Suitably, the tag isunique.

The coding part of the eventually formed bifunctional complex willcontain all the codons. The sequence of each of the codons is used todecipher the structure of the reactants that have participated in theformation of the displayed molecule, i.e. the reaction product. Theorder of the codons can also be used to determine the order ofincorporation of the reactants. This may be of particular interest whena linear polymer is formed, because the exact sequence of the polymercan be determined by decoding the encoding sequence. Usually, tofacilitate the decoding step, a constant or binding region istransferred to the bifunctional complex together with the codon. Theconstant region may contain information about the position of therelated reactant in the synthesis pathway of the display molecule.

The invention also relates to a method for identifying a displaymolecule having a preselected property, comprising the steps of:subjecting the library produced according to the method indicated aboveto a condition, wherein a display molecule or a subset of displaymolecules having a predetermined property is partitioned from theremainder of the library, and identifying the display molecule(s) havinga preselected function by decoding the coding part of the complex.

The above method, generally referred to as selection, involves that alibrary is subjected to a condition in order to select display moleculeshaving a property which is responsive to this condition. The conditionmay involve the exposure of the library to a target. The bifunctionalcomplexes having an affinity towards this target may be partitioned formthe remainder of the library by removing non-binding complexes andsubsequent eluting under more stringent conditions the complexes thathave bound to the target. Alternatively, the coding part of thebifunctional complex can be cleaved from the display molecule after theremoval of non-binding complexes and the coding part may be recoveredand decoded to identify the display molecule.

It is possible to perform a single or several rounds of selectionagainst a specific target with a subsequently amplification of theselected variants. These obtained variants are then separately tested ina suitable assay. The selection condition can be stringent and specificto obtain binding molecules in one selection rounds. It may beadvantageously to perform the method using a single round of selectionbecause the number and diversity of the potential binders are largercompared to procedures using further selections where potential bindersmay be lost. In another embodiment the selection procedure involvesseveral round of selection using increasing stringency conditions.Between each selection an amplification of the selected complex may bedesirable.

The coding part can be amplified using PCR with primers generating twounique cut-sites. These cut-sites can be used for multimerization of thecoding region by cloning into a suitable vector for sequencing. Thisapproach will allow simultaneously sequencing of many encoding regions.Alternatively, the PCR product is directly cloned into a suitable vectorusing for example TA cloning. In still another approach the identity ofthe display molecule is established by applying the PCR product to asuitable microarray.

It is within the capability of the skilled person in the art toconstruct the desired design of an oligonucleotide. When a specificannealing temperature is desired it is a standard procedure to suggestappropriate compositions of nucleic acid monomers and the lengththereof. The construction of an appropriate design may be assisted bysoftware, such as Vector NTI Suite or the public database at theinternet address .http://www.nwfsc.noaa.gov/protocols/oligoTMcalc. Theconditions which allow hybridisation of two oligonucleotides areinfluenced by a number of factors including temperature, saltconcentration, type of buffer, and acidity. It is within thecapabilities of the person skilled in the art to select appropriateconditions to ensure that the contacting between two oligonucleotides isperformed at hybridisation conditions. The temperature at which twosingle stranded oligonucleotides forms a duplex is referred to as theannealing temperature or the melting temperature. The melting curve isusually not sharp indicating that the annealing occurs over atemperature.

The present invention may be conducted in two basic modes. A first modeuses a reactant in which a codon or anti-codon covalently is connectedto the functional entity which it identifies. A second mode uses areactant which is not covalently attached to a codon or anti-codon. Thetag is provided at the priming site of the bifunctional complex by anentity separate from the reactant. When more than a single round iscarried out, the first and the second mode can be combined in any order.When a library of different bifunctional complexes is to be generated,the two modes are conducted in accordance with two different approaches.A library produced using the first mode can be conducted in a singlevessel, which herein will be referred to as a one-pot synthesis, whereasa library produced according to the second mode requires a split-and-mixsynthesis, i.e. the reaction and tag addition must be carried out inseparate compartments for each complex. In a certain embodiment of theinvention, one or more tags coding for two or more reactants,respectively, are provided prior to or subsequent to the reactioninvolving the two or more reactants and the chemical reaction site.

Mode 1:

The present invention relates in a first mode to a method for encodingthe identity of a chemical entity transferred to a bifunctional complex,said method comprising the steps of

a) providing a nascent bifunctional complex comprising a reactive groupand an oligonucleotide identifier region,b) providing a building block comprising an oligonucleotide sufficientcomplementary to the identifier region to allow for hybridisation, atransferable functional entity, and an anti-codon identifying thefunctional entity,c) mixing the nascent bifunctional complex and the building block underhybridisation conditions to form a hybridisation product,d) transferring the functional entity of the building block to thenascent bifunctional complex through a reaction involving the reactivegroup of the nascent bifunctional complex, ande) enzymatically extending the oligonucleotide identifier region toobtain a codon attached to the bifunctional complex having received thechemical entity.

The method of the invention involves the incorporation of a codon forthe functional entity transferred to the complex. The incorporation ofthe codon is performed by extending over an anticodon of the buildingblock using an appropriate enzyme, i.e. an enzyme active on nucleicacids. The transcription of the encoding region can be accomplished byan enzyme, such as a polymerase or a ligase. In general, it is preferredto use enzymes which are specific toward the substrate and theend-product to obtain an as accurate as possible transcription of theanti-codon. A high degree of specificity is generally available fornucleic acid active enzymes because a non-specific activity coulddestroy the ability of the living cells to survive. Especially preferredenzymes according to the present invention are polymerases withproof-reading activity for accurate encoding but preservation of theupstream nucleobases.

The enzymatic extension may occur subsequent to or simultaneously withthe transfer of the functional entity or even prior to the transfer.However, in general it is preferred to perform the extension stepsubsequent to the transfer step to avoid any possible interactionbetween the enzyme and the functional entity.

As the enzyme will perform extension only when the identifier region andthe complementing identifier region has hybridised to each other to forma double helix, it is secured that the functional entity and thereactive group has been in close proximity when the complex is providedwith a codon. Compared to the hybridisation method previously suggested,the present invention has the advantage that complexes provided withfunctional entities through a non-directed reaction will not be providedwith a codon. Thus, false positive molecules may easily be detected dueto the absence of a codon.

The invention also relates to a method for obtaining a bifunctionalcomplex composed of a display molecule part and a coding part, whereinthe method for encoding the identity of a chemical entity transferred toa bifunctional complex further comprises step f) separating thecomponents of the hybridisation product and recovering the complex.

The invention may be performed by transferring only a single functionalentity and the corresponding codon to the nascent bifunctional complex.However, in general it is preferred to build a display molecule composedof two of more functional entities. Thus, in a preferred aspect of theinvention a method is devised for obtaining a bifunctional complexcomposed of a display molecule part and a coding part, said displaymolecule part being the reaction product of functional entities and thereactive group of the initial complex, wherein steps c) to f) arerepeated as appropriate. In the final cycle of the preparation of thebifunctional complex, step f) may be dispensed with, notably in cases inwhich a double stranded identifier oligonucleotide is obtained because adouble stranded nucleic acid usually is more stable compared to acorresponding single stranded oligonucleotide. The identifieroligonucleotide may also become double stranded by an extension processin which a primer is annealed to the 3″end of the oligonucleotide andextended using a suitable polymerase. The double strandness may be anadvantage during subsequent selection processes because a singlestranded nucleic acid may perform interactions with a biological target,in a way similar to aptamers. In the repetition of the cycle, theproduced bifunctional complex in a previous cycle, i.e. a nascentbifunctional complex that has received a functional entity and a codon,is used as the nascent bifunctional complex in the next cycle offunctional entity transfer and codon incorporation.

The oligonucleotides used according to the present method are of areasonable extent. Thus, the long pre-made templates suggested in theprior art (in WO 00/23458 it is suggested to use oligonucleotides of atleast 220 and preferably 420 nucleotides) are generally avoided.

The invention also relates to a method for generating a library ofbifunctional complexes, comprising the steps of:

a) providing one or more different nascent bifunctional complexescomprising a reactive group and an oligonucleotide identifier region,b) providing a plurality of different building blocks, each comprisingan oligonucleotide sufficient complementary to an identifier region toallow for hybridisation, a transferable functional entity, and ananti-codon identifying the functional entity,c) mixing nascent bifunctional complexes and plurality of buildingblocks under hybridisation conditions to form hybridisation products,d) transferring functional entities of the building blocks to thenascent bifunctional complexes through a reaction involving the reactivegroup of the nascent bifunctional complex,e) enzymatically extending the oligonucleotide identifier regions toobtain codons attached to the bifunctional complexes having received thechemical entities,f) separating the components of the hybridisation products andrecovering the complexes,g) repeating steps c) to f) one or more times, as appropriate.

A disadvantage associated with the hybridisation technique suggested inthe prior art becomes apparent when the formation of libraries areconsidered. Even though two double stranded oligonucleotides have thesame number of nucleotides it is by no means ensured that they willpossess the same melting temperature. This is at least partly due to thefact that different number of hydrogen bondings are involved fordifferent base pairs (the C-G pair involves three hydrogen bondings andthe A-T base pair involves two hydrogen bondings). Thus, establishing atemperature for the annealing of various building blocks to a templatewill be a compromise between avoiding mismatching and ensuringsufficient annealing. The present invention aims at avoiding thisdisadvantage by providing, in a preferred embodiment of the invention,an identifier region having a similar affinity towards all buildingblocks.

In the event, more than one identifier sequence is used, e.g. when morethan one kind of reactive group or scaffolds are present, a buildingblock occasionally may be mis-annealed thereto. However, the transferredfunctional entity will actually be correctly encoded on the complexthrough the extension process. This approach resembles the arrangementNature is using: Allowing mis-incorporation of bases at the DNA level(compare to mismatch annealing of building blocks) to obtaindiversification but insisting on correct encoding for the phenotype(compare to the extension of the right codon on the complex).

The annealing between the identifier and the building block can eitherbe a random process or be guided by the sequences in the identifierregion and the complementing identifier region. A random process can beachieved by using the same sequence in all identifier regions and thecomplementing identifier regions. Thus a mixture of identifiers andbuilding blocks will anneal randomly or simi-randomly and create uniquecombinations of functional entities. Alternatively, a random orsimi-random process can be achieved by using universal bases atpositions of the building block opposing nucleobases of the identifierthat codes for the identity of a particular scaffold or reactive group.The sequences of the identifier oligonucleotides and the building blockoligonucleotides may be optimized such that it is assured that thesequences in a library involved in the annealing process will assembleat an equal degree of annealing regardless of which functional entitythat is attached to the building block. Thus, there will be no ordiminished bias in the selection procedure due to different annealingproperties for specific building blocks. In addition, the similaritiesin the annealing process in each annealing step and for eachhybridisation product in a library will make sure the functional entityis presented equally for the reactive group/scaffold. This will provideoptimal conditions for the transfer step.

The nascent bifunctional complex comprises an oligonucleotide identifierregion and a reactive group. The reactive group may be connected to theoligonucleotide through a cleavable linker allowing for the separationof the final reaction product from the oligonucleotide. A singlereactive group may be present or multiple reactive groups may be presentas a part of a scaffold. The scaffold may be attached to theoligonucleotide through a cleavable linker to allow for subsequentseparation of the reacted scaffold. The reactive groups may be selectedfrom any groups capable of receiving a functional entity. Examples ofsuitable reactive groups include amine, carboxylic, thio, and hydroxylgroups. Furthermore, the reactive group of the nascent bifunctionalcomplex may be in a pro-form that has to be activated before the methodof the invention is initiated. A nascent bifunctional complex is alsoreferred to as a growing complex and specifies an initial orintermediate complex to be further processed according to the presentinvention.

The number of nucleotides in the identifier region of the identifiermolecule is determined from how strong and specific the annealing shouldbe between the identifier and building block. A stronger and morespecific annealing process is generally obtained with a longernucleotide sequence. Normally about 10-20 nucleotides is sufficient toachieve specific and efficient annealing. However, in some aspects ofthe invention the range can be from 2-1000, most preferably between15-30 nucleotides.

The identifier region may in certain embodiments comprise informationabout the identity of the reactive group or the scaffold of the nascentbifunctional complex. Such scaffold codon is generally at a positiondistanced from the scaffold to allow for the formation of a stabledouble helix at the part comprising the functional entity to betransferred and the scaffold. The scaffold codon may have any length butis generally selected with the same length as the codons specifying thefunctional entities. The rear part of the identifier region is generallyprovided with a constant or binding sequence. The binding sequence whenannealed to a suitable part of the building block provides for asubstrate for the enzyme to perform the extension.

The building block comprises an oligonucleotide sufficient complementaryto at least a part of the identifier region to allow for hybridisation.The oligonucleotide of the building block may not completely becomplementary to the identifier, that is, one or more mismatches may beallowed but it must be assured that the building block is able to annealto the identifier region. For the sake of simplicity, the part of thebuilding block oligonucleotide capable of annealing to the identifierwill be referred to as the complementing identifier region. In thepresent description with claims, the term hybridisation is to beunderstood as the process of attaching two single strandedoligonucleotides to each other such that a hybridisation product isformed.

The building block comprises also an anticodon region made ofoligonucleotides. The anti-codon identifies the identity of thefunctional entity of the building block. In a certain embodiment, thesame anticodon is used to code for several different functionalentities. In a subsequent identification step, the structure of thedisplay molecule can be deduced taking advantage of the knowledge ofdifferent attachment chemistries, steric hindrance, deprotection oforthogonal protection groups, etc. In another embodiment, the sameanti-codon is used for a group of function entities having a commonproperty, such as a lipophilic nature, a certain attachment chemistryetc. In a preferred embodiment, however, the anti-codon is unique i.e. asimilar combination of nucleotides does not appear on another buildingblock carrying another functional entity. In a practical approach, for aspecific functional entity, only a single combination of nucleotides isused. In some aspects of the invention, it may be advantageous to useseveral anti-codons for the same functional entity, much in the same wayas Nature uses up to six different anti-codons for a single amino acid.The two or more anti-codons identifying the same functional entity maycarry further information related to different reaction conditions.

The individual anti-codons may be distinguished from another anti-codonin the library by only a single nucleotide. However, to facilitate asubsequent decoding process it is in general desired to have two or moremismatches between a particular anticodon and any other anti-codonappearing on the various building blocks. As an example, if acodon/anticodon length of 5 nucleotides is selected, more than 100nucleotide combinations exist in which two or more mismatches appear.For a certain number of nucleotides in the codon, it is generallydesired to optimize the number of mismatches between a particularcodon/anticodon relative to any other codon/anticodon appearing in thelibrary.

The coupling of the functional entity to the complementary identifierregion can be done with suitable coupling reactions. Any couplingreaction or combination of such reactions known in the art can be usedas appropriate as readily recognized by those skilled in the art. Thefunctional entity linked to the complementary identifier region is amolecule, which preferably comprises at least one reactive group thatallows linkage to the reactive group of the identifier.

The sequence of the anticodon identifies the functional entity attachedin the same building block. This anticodon sequence is either directlyincluded in the building block sequence or is attached to a pre-existingbuilding block using a polymerase or a ligase for example. In a certainembodiment, as disclosed in detail in example 7, complementingidentifier regions, termed carrier oligos in the example, are initiallyloaded with the various functional entities. Each of the loaded carrieroligos is subsequently ligated to an anti-codon oligo using a splintoligo to assemble the two oligonucleotides. The ligation reaction servesto connect the functional entity to be transferred with an anticodonspecifying the structure of the functional entity. The anti-codon oligomay be designed in various ways. Normally, a region that allows for theannealing of the splint is included in the design. However, some ligaseslike the T4 RNA ligase, does not require a stretch of double strandedDNA. Therefore, the splint and the part of the anti-codon oligoannealing to the splint can be dispensed with in some embodiments. Inthe event the identifier region comprises a codon coding for theidentity of the scaffold, the anti-codon oligo comprises a stretch ofuniversal bases, like inosines. The universal bases may be dispensedwith if a region complementing a binding region on the identifier regionis included downstream. The latter embodiment normally will entail thata part of the identifier loops out. The complementing binding region isnormally selected such that a polymerase is capable of recognizing aformed double helix with a binding region of the nascent bifunctionalmolecule as a substrate. The anti-codon is suitably positioned at the 5′side of the complementing binding region so it can be transferred to thenascent complex by an extension reaction. Suitably, the complementingbinding region is designed such that it is possible to identify theposition of the particular codon in the sequence of codons appearing onthe eventual bifunctional complex.

The anticodon sequence is transcribed to the identifier through anextension process to form the codon on the identifier molecule. This maybe carried out by any state of the art method including, but not limitedto, a polymerase extension reaction. A polymerase extension reactionusually requires the presence of sufficient polymerase activity togetherwith each of the four natural nucleotide tri-phosphates (ATP, CTP, GTP,and TTP) in a suitable buffer. Thus, the sequence of a particularanticodon is only transferred to the identifier as a codon when thebuilding block and the identifier molecule has annealed and allowreaction to take place between the functional entity and the recipientreactive group.

The four natural nucleotides can encode for 4^(N) variants where N isthe length of the codon. For example, if the unique codon is 5nucleotides in length, the number of possible encoding for differentfunctional entities is 1024. The codons can also be design using asub-set of the four natural nucleotides in each position. This can beuseful in combination with the use of universal nucleobases. Theanticodon in each building block is coding for the functional entity inthe same building block. This sequence may in an aspect of the inventionbe incorporated by PCR of the complementing identifier region with afunctional entity primer and an anticodon primer.

The functional entity of the building block serves the function of beinga precursor for the structural entity eventually incorporated into thedisplayed molecule. Therefore, when in the present application withclaims it is stated that a functional entity is transferred to a nascentbifunctional complex it is to be understood that not necessarily all theatoms of the original functional entity is to be found in the eventuallyformed display molecule. Also, as a consequence of the reactionsinvolved in the connection, the structure of the functional entity canbe changed when it appears on the nascent display molecule. Especially,the cleavage resulting in the release of the functional entity maygenerate a reactive group which in a subsequent step can participate inthe formation of a connection between a nascent display molecule and afunctional entity.

The functional entity of the building block preferably comprises atleast one reactive group capable of participating in a reaction whichresults in a connection between the functional entity of the buildingblock and the identifier carrying the reactive group. The number ofreactive groups which appear on the functional entity is suitably one toten. A functional entity featuring only one reactive group is used i.a.in the end positions of polymers or scaffolds, whereas functionalentities having two reactive groups are suitable for the formation ofthe body part of a polymer or scaffolds capable of being reactedfurther. Two or more reactive groups intended for the formation ofconnections, are typically present on scaffolds. A scaffold is a corestructure, which forms the basis for the creation of multiple variants.The variant forms of the scaffold are typically formed through reactionof reactive groups of the scaffold with reactive groups of otherfunctional entities, optionally mediated by fill-in groups or catalysts.The functional entities to be connected to the scaffold may contain one,two or several reactive groups able to form connections. Examples ofscaffold include steroids, hydantions, benzodiazepines, etc.

The reactive group of the building block may be capable of forming adirect connection to a reactive group of the identifier or the reactivegroup of the building block may be capable of forming a connection to areactive group of the identifier through a bridging fill-in group. It isto be understood that not all the atoms of a reactive group arenecessarily maintained in the connection formed. Rather, the reactivegroups are to be regarded as precursors for the structure of theconnection.

After or simultaneously with the formation of the connection a cleavageis performed to transfer the functional entity to the identifier. Thecleavage can be performed in any appropriate way. In an aspect of theinvention the cleavage involves usage of a reagent or and enzyme. Thecleavage results in a transfer of the functional entity to the nascentbifunctional complex or in a transfer of the complex to the functionalentity of the building block. In some cases it may be advantageous tointroduce new chemical groups as a consequence of linker cleavage. Thenew chemical groups may be used for further reaction in a subsequentcycle, either directly or after having been activated. In other cases itis desirable that no trace of the linker remains after the cleavage.

In another aspect, the connection and the cleavage is conducted as asimultaneous reaction, i.e. either the functional entity of the buildingblock or the nascent display molecule is a leaving group of thereaction. In some aspects of the invention, it is preferred to designthe system such that the connection and the cleavage occursimultaneously because this will reduce the number of steps and thecomplexity. The simultaneous connection and cleavage can also bedesigned such that either no trace of the linker remains or such that anew chemical group for further reaction is introduced, as describedabove. In other aspects of the invention, it is preferred to conductseparate cross-linkage and cleavage steps because the stepwise approachallows for mastering each sub steps and for a reduction in thelikelihood for non-specific transfer.

Preferably, at least one linker remains intact after the cleavage step.The at least one linker will link the nascent display molecule to theencoding region. In case the method essentially involves the transfer offunctional entities to a scaffold or an evolving polymer, the eventuallyscaffolded molecule or the polymer may be attached with a selectivelycleavable linker. The selectively cleavable linker is designed such thatit is not cleaved under conditions which result in a transfer of thefunctional entity to the nascent template-directed molecule.

The cleavable linkers may be selected from a large plethora of chemicalstructures. Examples of linkers includes, but are not limited to,linkers having an enzymatic cleavage site, linkers comprising a chemicaldegradable component, and linkers cleavable by electromagneticradiation. Cleavable linkers of particular interest are currentlylinkers that can be cleaved by light. A suitable example includes ano-nitro benzyl group positioned between the display molecule and theidentifier region.

The building blocks used in the method according to the presentinvention may be designed in accordance with the particular entitiesinvolved in the building block. As an example, the anti-codon may beattached to the complementing identifier region with a polyethyleneglycol (PEG) linker and the functional entity may be directly attachedto said complementing identifier region. In another and preferredexample, the anti-codon, complementing identifier region and thefunctional entity is a contiguous linear oligonucleotide. In a certainembodiment of the invention, the building block is designed such that apart of the identifier loops out. The loop out of the identifier usuallyoccurs because the building block oligo does not anneal to the entirelength of the identifier. Usually, the building block is designed suchthat it is able to anneal to at least the identifier region of thebifunctional complex and to a binding region at the rear part of theidentifier. The complementing identifier region and the anticodon may bedirectly connected through a single linkage, connected through a PEGlinker of a suitable length, or a sequence of nucleobases which may ormay not comprise nucleobases complementing the various codons andbinding region on the identifier. In a certain embodiment of theinvention, the building block is designed only to anneal to a bindingregion, usually at an end of the identifier opposing the end havingattached the display molecule. In an aspect of the invention thebuilding block and/or the nascent identifier are composed of two or moreseparate nucleotides, which are able to hybridise to each other to formthe hybridisation complex. The gaps between the oligonucleotides may befilled with suitable nucleotide using an appropriate enzyme activity,such as a polymerase and a ligase, to produce a coherent identifier andor building block.

The attachment of the functional entity to the complementing identifierregion is usually conducted through a linker. Preferably the linkerconnects the functional entity with the complementing identifier regionat a terminal nucleotide or a nucleotide 1 or two nucleotides down theoligonucleotide. The attachment of the functional entity can be at anyentity available for attachment, i.e. the functional entity can beattached to a nucleotide of the oligonucleotide at the nucleobase, orthe back bone. In general, it is preferred to attach the functionalentity at the phosphor of the internucleoside linkage or at thenucleobase.

In a certain aspect of the invention, the reactive group of thefunctional entity is attached to the linker oligonucleotide. Thereactive group is preferably of a type which is able to create aconnection to the nascent display molecule by either direct reactionbetween the respective reactive groups or by using a suitable fill-ingroup. The reactive group coupling the functional entity with the linkeris preferably cleaved simultaneously with the establishment of theconnection. The functional entity may in some cases contain a secondreactive group able to be involved in the formation of a connection in asubsequent cycle. The second reactive group may be of a type which needsactivation before it is capable of participating in the formation of aconnection.

In the event two or more functional entities are to be transferred tothe complex, the codons may be separated by a constant region or abinding region. One function of the binding region may be to establish aplatform at which the polymerase can bind. Depending on the encodedmolecule formed, the identifier may comprise further codons, such as 3,4, 5, or more codons. Each of the further codons may be separated by asuitable binding region. Preferably, all or at least a majority of thecodons of the identifier are separated from a neighbouring codon by abinding sequence. The binding region may have any suitable number ofnucleotides, e.g. 1 to 20.

The binding region, if present, may serve various purposes besidesserving as a substrate for an enzyme. In one setup of the invention, thebinding region identifies the position of the codon. Usually, thebinding region either upstream or downstream of a codon comprisesinformation which allows determination of the position of the codon. Inanother setup, the binding regions have alternating sequences, allowingfor addition of building blocks from two pools in the formation of thelibrary. Moreover, the binding region may adjust the annealingtemperature to a desired level.

A binding region with high affinity can be provided by incorporation ofone or more nucleobases forming three hydrogen bonds to a cognatenucleobase. Examples of nucleobases having this property are guanine andcytosine. Alternatively, or in addition, the binding region may besubjected to backbone modification. Several backbone modificationsprovides for higher affinity, such as 2′-O-methyl substitution of theribose moiety, peptide nucleic acids (PNA), and 2′-4′ O-methylenecyclisation of the ribose moiety, also referred to as LNA (LockedNucleic Acid).

The identifier may comprise flanking regions around the codons. Theflanking region can encompass a signal group, such as a fluorophor or aradio active group to allow for detection of the presence or absence ofa complex or the flanking region may comprise a label that may bedetected, such as biotin. When the identifier comprises a biotin moiety,the identifier may easily be recovered.

The flanking regions can also serve as priming sites for amplificationreactions, such as PCR. Usually, the last cycle in the formation of thebifunctional complex includes the incorporation of a priming site. Theidentifier region of the bifunctional complex is usually used foranother priming site, thereby allowing for PCR amplification of thecoding region of the bifunctional complex.

It is to be understood that when the term identifier is used in thepresent description and claims, the identifier may be in the sense orthe anti-sense format, i.e. the identifier can comprise a sequence ofcodons which actually codes for the molecule or can be a sequencecomplementary thereto. Moreover, the identifier may be single-strandedor double-stranded, as appropriate.

The design of the part of the complementing identifier region or thebuilding block oligonucleotide in general which comprises one or moreanti-codons preceding the active anti-codon can be random or simi-randomand one or more mismatches with the identifier region may be allowed.However, especially when a library is contemplated, it may beadvantageous to incorporate in a region complementing a preceding codonone or more non-specific base-pairing nucleobases. Non-specificbase-pairing nucleobases are bases which, when attached to a backbone,are able to pair with at least two of the five naturally occurringnucleobases (C, T, G, A, and U). Preferably, the base pairing betweenthe two or more natural nucleobases and the non-specificallybase-pairing nucleobase occur essentially iso-enegically, i.e. the bondsformed have a strength of the same order. The term “non-specificallybase-pairing nucleobase” is used herein interchangeably with the term“universal base”.

In natural tRNA, the nucleobase inosine is found. Inosine has theability to hybridise non-specifically with three of the nucleobases,i.e. cytosine, thymine, and adenine. Inosine and examples of othersynthetic compounds having the same ability of non-specificallybase-pairing with natural nucleobases are depicted below

Examples of Universal Bases

The use of universal bases in the present method has an advantage in thegeneration of a library because the nucleobases of previouslytransferred codons can be matched with universal bases on thecomplementing region of the building block. The complementing of a spentcodon with a sequence of universal bases allows for the use of the samebuilding block for a variety of growing bifunctional complexes.

The encoding by extension principle can also be used using athree-strand procedure. Each step involves a library of assemblyplatform molecules hybridised to a functional entity carrier (FIG. 7).The assembly platform comprise a fixed sequence (complementingidentifier region) that binds equally well to all or a subset ofidentifier molecule through the identifier region. Alternatively, thiscomplementing identifier sequence can also be random or simi-random toincrease the diversity of the library as this would allow for the use ofdifferent scaffold molecules. The assembly platform also contains aunique anticodon region with a specific sequence. This specific sequencewill anneal to the unique codon region in the carrier, thus forming abuilding block in which the transferable functional entity is coupled toa unique anti-codon by hybridisation. The sequence of the uniqueanticodon and the unique anticodon region is linked allowing a directcoupling between these two sequences. This coupling is for exampleobtained when the assembly platform is synthesized.

The unique anticodon can either be identical to the unique anticodonregion or a shorter or longer sequence. However, a prerequisite thoughis that these two sequences (the unique anticodon and the uniqueanticodon region) are linked to each other, e.g. through thecomplementing identifier region and, optionally, the connection region.The sequence of the unique anticodon can be used to decode the uniqueanticodon region. This will obtain the unique codon region which codesfor the functional entity. The connecting region is optionally asequence that can be varied to obtain optimal reactivity betweenfunctional entity and the attachment entity. If polymers are createdusing this system, the connecting region could be extended through theassembling cycles.

The formation of identifier-displayed molecules by the three-strandassembly principle is performed in sequential steps. Each individualstep involves annealing of the carrier and the identifier molecules tothe assembly platform. After the annealing step, two important eventstake place: 1) the reaction between the attachment entity and thefunctional entity to accomplish transfer of the functional entity to theidentifier molecule, and 2) the extension of the unique codon sequenceinto the identifier molecule using the unique anticodon sequence on theassembly platform as the reading sequence.

The formation of a library of bifunctional complexes according to theinvention can be performed using a solid support for the platformmolecule as shown in FIGS. 9 and 10. This allow a sequential transferwhere each library of assembly platform molecules, with differentaddition of the non-coding region and complementing binding regiondependent of which specific step, is immobilized in separate vials and alibrary of identifier and building block molecules is supplied. Afterthe annealing-reaction/transfer-extension steps, the library is removed(e.g. with elevated temperature) and transferred to another vial with animmobilized assembly platform library (with an additional non-coding andcomplementing binding region) to allow the next step in the process.

Mode 2:

The present invention discloses in a second mode of the invention, amethod for generating a complex comprising a display molecule part and acoding part, wherein a nascent bifunctional complex comprising achemical reaction site and a priming site for enzymatic addition of atag is reacted at the chemical reaction site with one or more reactantsand provided at the priming site with respective tags identifying theone or more reactants using one or more enzymes.

The lack of a covalent link between the reactive part and the codingpart of the building block implies that a library is to be produced by asplit-and-mix strategy. In a first step a nascent bifunctional complexis dispensed in one or more separate compartment and subsequentlyexposed to a reactant in each compartment, which reacts at the chemicalreaction site, and an agent which provides the tag identifying saidreactant at the priming site. The agent providing the tag includes anenzyme and a substrate therefore. In a certain embodiment of theinvention, the tag is provided by extending over an anti-codon using apolymerase. In another embodiment of the invention, the tag is providedat the priming site by ligation of a codon oligonucleotide, which holdsinformation as to the identity of the reactant.

When the enzyme is a polymerase, the substrate is usually a blend oftriphosphate nucleotides selected from the group comprising dATP, dGTP,dTTP, dCTP, rATP, rGTP, rTTP, rCTP, rUTP. Substrates for ligases areoligo- and polynucleotides, i.e. nucleic acids comprising two or morenucleotides. An enzymatic ligation may be performed in a single ordouble stranded fashion. When a single stranded ligation is performed, a3′ OH group of a first nucleic acid is ligated to a 5′ phosphate groupof a second nucleic acid.

A double stranded ligation uses a third oligonucleotide complementing apart of the 3′ end and 5′ end of the first and second nucleic acid toassist in the ligation. Generally, it is preferred to perform a doublestranded ligation.

In some embodiments of the invention, a combination of polymerasetranscription and ligational coupling is used. As an example, a gap inan otherwise double stranded nucleic acid may be filled-in by apolymerase and a ligase can ligate the extension product to the upstreamoligonucleotide to produce a wholly double stranded nucleic acid.

Mode 2 is conducted in separate compartments for each reaction, asdiscussed above. Thus, the addition of a tag occurs without competingnucleic acids present and the likelihood of cross-encoding is reducedconsiderable. The enzymatic addition of a tag may occur prior to,subsequent to, or simultaneous with the reaction. In some aspects of theinvention, it is preferred to add the tag to the nascent bifunctionalcomplex prior to the reaction, because it may be preferable to applyconditions for the reaction which are different form the conditions usedby the enzyme. Generally, enzyme reactions are conducted in aqueousmedia, whereas the reaction between the reactant and the chemicalreaction site for certain reactions is favoured by an organic solvent.An appropriate approach to obtain suitable condition for both reactionsis to conduct the enzyme reaction in an aqueous media, lyophilize andsubsequent dissolve or disperse in a media suitable of the reaction atthe chemical reactive site to take place. In an alternative approach,the lyophilization step may be dispensed with as the appropriatereaction condition can be obtained by adding a solvent to the aqueousmedia. The solvent may be miscible with the aqueous media to produce ahomogeneous reaction media or immiscible to produce a bi-phasic media.

The reactant according to the second mode may be a free reactant or azipper building block. A free reactant is not attached to a codeidentifying another part of the reactant. In most cases, a free reactantcomprises a chemical structure comprising one, two or more reactivegroups, which can react with the chemical reaction site. A zipperbuilding block is a functional entity which is attached to a chemicalentity that binds in the vicinity of the chemical reaction site. Thebinding chemical entity may be an oligonucleotide which hybridises to alinking moiety of the nascent bifunctional complex prior to thereaction. The hybridisation event will increase the proximity betweenthe functional entity and the chemical reaction site, thereby reducingthe possibility of side reactions and promote the reaction due to a highlocal concentration.

The nascent bifunctional complex is constructed having the encodingmethod in mind. Thus, if a polymerase is used for the encoding, a regionof hybridisation is usually provided in the linker moiety. The region ofhybridisation will allow for a binding region of a complementingoligonucleotide comprising an anti-codon to hybridise to the nascentbifunctional complex. The binding region serves as a binding site for apolymerase, which then may produce an extension product using theanti-codon oligonucleotide as template. When a ligase is used for theencoding, the priming site of the nascent bifunctional complex comprisesone or more nucleotides which the ligase may consider as a substrate. Ina single stranded ligation an oligonucleotide present in the media andbearing information as to the identity of the reactive group will beligated to the nascent bifunctional molecule. A double stranded ligationrequires the priming site of the nascent bifunctional complex to be ableto hybridise to a complementing oligonucleotide prior to ligation.Suitably, the priming site comprises one, two, or more nucleotides, towhich a complementing oligonucleotide can hybridise. The complementingoligonucleotide hybridise in the other end to the codon oligonucleotide,which holds the information of a particular reactant.

The linker moiety of the nascent bifunctional complex may compriseinformation relating to the identity of the chemical reaction site. Inan applicable approach, the linker moiety comprises a codon informativeof the identity of the chemical reaction site.

The oligonucleotides bearing the information on the pertinent reactant,may, apart from the combination of nucleotides identifying the reactant,comprise flanking regions. The flanking regions may serve as bindingregions capable of hybridising to the nascent bifunctional complex. Thebinding region may be designed so as to hybridise promiscuous to morethan a single nascent bifunctional complex. Alternatively, the bindingregion on the coding oligonucleotide is capable of being ligated to abinding region the nascent bifunctional complex using a splintoligonucleotide as mediator.

The invention may be performed by reacting a single reactant with thenascent bifunctional complex and add the corresponding tag. However, ingeneral it is preferred to build a display molecule comprising thereaction product of two of more reactants.

Thus, in a certain aspect of the invention a method is devised forobtaining a bifunctional complex composed of a display molecule part anda coding part, said display molecule part being the reaction product ofreactants and the chemical reaction site of the initial complex. In anaspect of the invention, two alternating parallel syntheses areperformed so that the tag is enzymatical linked to the nascentbifunctional complex in parallel with a reaction between a chemicalreaction site and a reactant. In each round the addition of the tag isfollowed or preceded by a reaction between reactant and the chemicalreaction site. In each subsequent round of parallel syntheses thereaction product of the previous reactions serves as the chemicalreaction site and the last-incorporated tag provides for a priming sitewhich allows for the enzymatical addition a tag. In other aspects of theinvention, two or more tags are provided prior to or subsequent toreaction with the respective reactants.

The coding part comprising all the tags may be transformed to a doublestranded form by an extension process in which a primer is annealed tothe 3′ end of the oligonucleotide and extended using a suitablepolymerase. The double strandness may be an advantage during subsequentselection processes because a single stranded nucleic acid may performinteractions with a biological target in a way similar to aptamers.

In a certain aspect of mode 2 a method is devised for generating alibrary of bifunctional complexes comprising a display molecule part anda coding part. The method comprises the steps of providing in separatecompartments nascent bifunctional complexes, each comprising a chemicalreaction site and a priming site for enzymatic addition of a tag andperforming in any order reaction in each compartment between thechemical reaction site and one or more reactants, and addition of one ormore respective tags identifying the one or more reactants at thepriming site using one or more enzymes.

The nascent bifunctional complexes in each compartment may be identicalor different. In the event the nascent bifunctional complex differs atthe chemical reaction site, the nascent bifunctional complex suitablecomprises a codon identifying the structure of the chemical reactionsite. Similar, the reactants applied in each compartment may beidentical or different as the case may be. Also, the reaction conditionsin each compartment may be similar or different.

Usually, it is desired to react the complex with more than a singlereactant. In a certain aspect of the invention, the content of two ormore compartments are pooled together and subsequently split into anarray of compartments for a new round of reaction. Thus, in any roundsubsequent to the first round, the end product of a preceding round ofreaction is used as the nascent bifunctional complex to obtain a libraryof bifunctional complexes, in which each member of the library comprisesa reagent specific reaction product and respective tags which codes forthe identity of each of the reactants that have participated in theformation of the reaction product. Between each round of reaction thecontent of the compartments is in an aspect of the invention mixedtogether and split into compartments again. In other aspects of theinvention the content of a compartment is after having received a codonbut before a reaction has occurred divided into further compartments inwhich a further codon is received and a reaction occurs with the tworeactants that have been encoded. In another aspect of the invention,more than two codons are encoded before a reaction between chemicalreaction site and reactants are allowed to take place. In thealternative, two or more reactions are allowed to occur before anencoding with the respective tags is initiated.

The individual codons may be distinguished from another codon in thelibrary by only a single nucleotide. However, to facilitate a subsequentdecoding process it is in general desired to have two or moredifferences between a particular codon and any other codon. As anexample, if a codon/anticodon length of 5 nucleotides is selected, morethan 100 nucleotide combinations exist in which two or more differencesappear. For a certain number of nucleotides in the codon, it isgenerally desired to optimize the number of differences between aparticular codon/anticodon relative to any other codon/anticodonappearing in the library. An oligonucleotide codon may comprise anysuitable number of nucleotides, such as from 2 to 100, 3 to 50, 4 to 20or 5 to 15 nucleotides.

The reactant can be a free reactant or a zipper building block. Thereactant serves the function of being a precursor for the structuralentity eventually incorporated in to the displayed molecule part. Therestructure of a reactant may after reaction with a chemical reaction sitebecome changed in a subsequent round. In the event the reactant is azipper building block, a cleavage of the linkage between the functionalentity and the oligonucleotide is normally conducted after reaction. Anexception is in the final round, in which the cleavage can be dispensedwith. The cleavage can occur subsequent to or simultaneously with thereaction with the chemical reaction site. The cleavage may generate areactive group which in a subsequent step can participate in theformation of a connection between the nascent display molecule and areactant.

The free reactant or the functional entity of the zipper building blockpreferably comprises at least one reactive group capable ofparticipating in a reaction which results in a connection to thechemical reaction site of the nascent bifunctional molecule. The numberof reactive groups which appear on the free reactant and the functionalentity is suitably one to ten. A free reactant or a functional entityfeaturing only one reactive group is used i.a. in the end positions ofpolymers or scaffolds, whereas functional entities having two reactivegroups are suitable for the formation of the body part of a polymer orscaffolds capable of being reacted further. Two or more reactive groupsintended for the formation of connections, are typically present onscaffolds. A scaffold is a core structure, which forms the basis for thecreation of multiple variants. The variant forms of the scaffold aretypically formed through reaction of reactive groups of the scaffoldwith reactive groups of other reactants, optionally mediated by fill-ingroups or catalysts. The functional entities or free reactants to beconnected to the scaffold may contain one, two or several reactivegroups able to form connections. Examples of scaffolds include steroids,hydantions, benzodiazepines, etc.

The reactive group of the free reactant or the functional entityattached to a nucleic acid comprising a zipper region, i.e. a regionpromiscuously binding to a linking moiety of the nascent bifunctionalcomplex, may be capable of forming a direct connection to a reactivegroups of the chemical reactive site or the reactant may be capable offorming a connection to a reactive group of the chemical reactive sitethrough a bridging fill-in group. It is to be understood that not allthe atoms of the reactive groups are necessarily maintained in theconnection formed. Rather the reactive groups are to be regarded asprecursors for the structure of the connection.

When a zipper building block is used, a cleavage may be performed afteror simultaneously with the formation of the connection between thechemical reaction site and the functional entity. The cleavage can beperformed in any appropriate way. In an aspect of the invention thecleavage involves usage of a reagent or enzyme. The cleavage results ina transfer of the functional entity to the nascent bifunctional complexor in a transfer of the complex to the functional entity of the zipperbuilding block. In some cases it may be advantageous to introduce newchemical groups as consequence of the cleavage. The new chemical groupsmay be used for further reaction in a subsequent cycle, either directlyor after having been activated. In other cases it s desirable that notrace of the linker remains after the cleavage. In some aspects of theinvention it may not be desired to cleave on or more chemical bonds. Asan example, it may be desirable to maintain the connection between thezipper domain and the functional entity in the last round.

In some aspects of the invention, the connection and the cleavage isconducted as a simultaneous reaction, i.e. either the functional entityof the zipper building block or the chemical reactive site of thenascent bifunctional complex is a leaving group of the reaction. In someaspects of the invention, it is preferred to design the system such thatthe cleavage occurs simultaneously because this will reduce the numberof steps and the complexity. The simultaneous connection and cleavagecan also be designed such that either no trace of the linker remains orsuch that a new chemical group for further reaction is introduced, asdescribed above. In other aspects of the invention, it is preferred toconduct separate cross-linking and cleavage steps because the stepwiseapproach allows for mastering each sub step and for a reduction of thelikelihood of non-specific transfer.

The attachment of the functional entity to the oligonucleotidecomprising a zipping domain is usually conducted through a linker.Preferably the linker connects the functional entity with theoligonucleotide at a terminal nucleotide or a nucleotide 1 or twonucleotides down the oligonucleotide. The attachment of the functionalentity can be at any entity available for attachment, i.e. thefunctional entity can be attached to a nucleotide of the oligonucleotideat the nucleobase, or the back bone. In general, it is preferred toattach the functional entity at the phosphor of the internucleosidelinkage or at the nucleobase.

In a certain aspect of the invention, the reactive group of thefunctional entity is attached to the oligonucleotide, optionally througha suitable spacer. The reactive group is preferably of a type which isable to create a connection to the nascent display molecule by eitherdirect reaction between the respective reactive groups or by using asuitable fill-in group. The reactive group coupling the functionalentity with the oligonucleotide is preferably cleaved simultaneouslywith the establishment of the connection. The functional entity may insome cases contain a second reactive group able to be involved in theformation of a connection in a subsequent cycle. The second reactivegroup may be of a type which needs activation before it is capable ofparticipating in the formation of a connection.

Preferably at least one linker remains intact after the cleavage step.The at least one linker will link the display molecule to the codingpart, i.e. the part comprising the one or more tags identifying thevarious reactant that have participated in the formation of the displaymolecule. It may be desired to connect the display molecule part to thecoding part of the bifunctional complex through a space comprising aselectively cleavable linker. The selectively cleavable linker isdesigned such that it is not cleaved under conditions which result in atransfer of a function entity to the chemical reaction site.

The cleavable linkers may be selected from a large plethora of chemicalstructures. Examples of linkers includes, but are not limited to,linkers having an enzymatic cleavage site, linkers comprising a chemicaldegradable component, and linkers cleavable by electromagneticradiation. Cleavable linkers of particular interest are currentlylinkers that can be cleaved by light. A suitable example includes ano-nitro benzyl group positioned between the display molecule and thecoding part of the bifunctional complex.

In the event two or more reactants are reacted with the chemicalreactive site, the codons of the coding part may be separated by aconstant region or a binding region. One function of the binding regionmay be to establish a platform at which an enzyme, such as polymerase orligase can recognise as a substrate. Depending on the encoded moleculeformed, the identifier may comprise further codons, such as 3, 4, 5, ormore codons. Each of the further codons may be separated by a suitablebinding region. Preferably, all or at least a majority of the codons ofthe identifier are separated from a neighbouring codon by a bindingsequence. The binding region may have any suitable number ofnucleotides, e.g. 1 to 20.

The binding region, if present, may serve various purposes besidesserving as a substrate for an enzyme. In one setup of the invention, thebinding region identifies the position of the codon. Usually, thebinding region either upstream or downstream of a codon comprisesinformation which allows determination of the position of the codon. Inanother setup, the binding regions have alternating sequences, allowingfor addition of building blocks from two pools in the formation of thelibrary. Moreover, the binding region may adjust the annealingtemperature to a desired level.

A binding region with high affinity can be provided by incorporation ofone or more nucleobases forming three hydrogen bonds to a cognatenucleobase. Examples of nucleobases having this property are guanine andcytosine. Alternatively, or in addition, the binding region may besubjected to backbone modification. Several backbone modificationsprovides for higher affinity, such as 2′-O-methyl substitution of theribose moiety, peptide nucleic acids (PNA), and 2′-4′ O-methylenecyclisation of the ribose moiety, also referred to as LNA (LockedNucleic Acid).

The identifier may comprise flanking regions around the codons. Theflanking region can encompass a signal group, such as a fluorophor or aradio active group to allow for detection of the presence or absence ofa complex or the flanking region may comprise a label that may bedetected, such as biotin. When the identifier comprises a biotin moiety,the identifier may easily be recovered.

The flanking regions can also serve as priming sites for amplificationreactions, such as PCR. Usually, the last cycle in the formation of thebifunctional complex includes the incorporation of a priming site. Aregion of the bifunctional complex close to the display molecule, suchas a nucleic acid sequence between the display molecule and the codoncoding for the scaffold molecule, is usually used for another primingsite, thereby allowing for PCR amplification of the coding region of thebifunctional complex.

Combination of Mode 1 and Mode 2:

In a certain aspect of the invention, mode 1 and mode 2 described aboveis combined, i.e. different reactants are used in different rounds. Alsowithin mode 1 and mode 2 different building blocks may be used indifferent rounds.

In the formation of a library it may be advantageous to use acombination of a one-pot synthesis strategy (mode 1) and a split-and-mixstrategy (mode 2), because each of mode 1 and mode 2 has its virtues.The one-pot strategy offers the possibility of having the reactivegroups in close proximity prior to reaction, thus obtaining a high localconcentration and the convenience of having a single container. Thesplit- and mix strategy offers the possibility of having a free reactantand non-hybridising reaction conditions, providing for versatilereactions. It may be appropriate to refer to FIG. 15 in which varioussingle encoding enzymatic methods are shown. A split-and-mix synthesisstrategy is generally used for reactants not having a covalent linkbetween the reactant/functional entity and the codon/anti-codon, i.e.free reactants and zipper building blocks. A one-pot synthesis strategyis generally used for reactants in which a covalent link exist betweenthe functional entity and the codon/anti-codon identifying saidfunctional entity, i.e. the E2 building blocks, loop building blocks,and the N building blocks.

In a certain embodiment of the invention an intermediate library ofbifunctional complexes is generated using a one-pot synthesis strategy.This intermediate library is subsequently used for the generation of afinal library by a split-and-mix synthesis. The intermediate library maybe generated using a single round or multiple rounds of one-potsynthesis and the final library may be produced applying a single ormultiple rounds of split-and-mix. The use of a split-and-mix synthesisin the last round of library generation offers the possibility of usinga reaction media not compatible with maintenance of a hybridisation,e.g. high ionic strength or organic solvents, for the final reactant.

In another embodiment an intermediate library is produced using a splitand mix synthesis strategy. The intermediate library is used for thegeneration of a final library using a one-pot synthesis strategy. Theintermediate library may be produced using a single or multiple roundsof split-and-mix synthesis and the final library may be manufacturedapplying one or more rounds of one-pot synthesis. The one-pot synthesisin the final round provide for a close proximity between the growingencoded molecule and the functional entity. The close proximity resultsin a high local concentration promoting the reaction even for reactantshaving a relatively low tendency to react.

Multiple Encoding

Multiple encoding implies that two or more codons are provided in theidentifier prior to or subsequent to a reaction between the chemicalreactive site and two or more reactants. Multiple encoding has variousadvantages, such allowing a broader range of reactions possible, as manycompounds can only be synthesis by a three (or more) component reactionbecause an intermediate between the first reactant and the chemicalreactive site is not stable. Other advantages relates to the use oforganic solvents and the availability of two or more free reactants incertain embodiments.

Thus in a certain aspect of the invention, it relates to a method forobtaining a bifunctional complex comprising a display molecule part anda coding part, wherein the display molecule is obtained by reaction of achemical reactive site with two or more reactants and the coding partcomprises tag(s) identifying the reactants.

In a certain aspect of the invention, a first reactant forms anintermediate product upon reaction with the chemical reactive site and asecond reactant reacts with the intermediate product to obtain thedisplay molecule or a precursor thereof. In another aspect of theinvention, two or more reactants react with each other to form anintermediate product and the chemical reactive site reacts with thisintermediate product to obtain the display molecule or a precursorthereof. The intermediate product can be obtained by reacting the two ormore reactants separately and then in a subsequent step reacting theintermediate product with the chemical reactive site. Reacting thereactants in a separate step provide for the possibility of usingconditions the tags would not withstand. Thus, in case the coding partcomprises nucleic acids, the reaction between the reactant may beconducted at conditions that otherwise would degrade the nucleic acid.

The reactions may be carried out in accordance with the scheme shownbelow. The scheme shows an example in which the identifying tags for tworeactants and the chemical reactive site (scaffold) attached to thechemical reaction site are provided in separate compartments. Thecompartments are arranged in an array, such as a microtiter plate,allowing for any combination of the different acylating agents and thedifferent alkylating agents.

Starting Situation:

Alkylating agents Acylating agents A B C . . . 1 Tagx11-X Tagx12-XTagx13-X . . . 2 Tagx21-X Tagx22-X Tagx23-X . . . 3 Tagx31-X Tagx32-XTagx33-X . . . . . . . . . . . . . . . . . . X denotes a chemicalreaction site such as a scaffold.

The two reactants are either separately reacted with each other in anycombination or subsequently added to each compartment in accordance withthe tags of the coding part or the reactants may be added in any orderto each compartment to allow for a direct reaction. The scheme belowshows the result of the reaction.

Plate of Products

Alkylating agents Acylating agents A B C . . . 1 Tagx11-XA1 Tagx12-XB1Tagx13-XC1 . . . 2 Tagx21-XA2 Tagx22-XB2 Tagx23-XC2 . . . 3 Tagx31-XA3Tagx32-XB3 Tagx33-XC3 . . . . . . . . . . . . . . . . . .

As an example XA2 denotes display molecule XA2 in its final state, i.e.fully assembled from fragments X, A and 2.

The coding part comprising the two or more tags identifying thereactants, may be prepared in any suitable way either before or afterthe reaction. In one aspect of the invention, each of the coding partsare synthesised by standard phosphoramidite chemistry. In another aspectthe tags are pre-prepared and assembled into the final coding part bychemical or enzymatic ligation.

Various possibilities for chemical ligation exist. Suitable examplesinclude that

a) a first oligonucleotide end comprises a 3′-OH group and the secondoligonucleotide end comprises a 5′-phosphor-2-imidazole group. Whenreacted a phosphodiester internucleoside linkage is formed,b) a first oligonucleotide end comprising a phosphoimidazolide group andthe 3′-end and a phosphoimidazolide group at the 5′-and. When reactedtogether a phosphodiester internucleoside linkage is formed,c) a first oligonucleotide end comprising a 3′-phosphorothioate groupand a second oligonucleotide comprising a 5′-iodine. When the two groupsare reacted a 3′-O—P(═O)(OH)—S-5′ internucleoside linkage is formed, andd) a first oligonucleotide end comprising a 3′-phosphorothioate groupand a second oligonucleotide comprising a 5′-tosylate. When reacted a3′-O—P(═O)(OH)—S-5′ internucleoside linkage is formed.

Suitably, the tags operatively are joined together, so that as to allowa nucleic acid active enzyme to recognize the ligation area assubstrate. Notably, in a preferred embodiment, the ligation is performedso as to allow a polymerase to recognise the ligated strand as atemplate. Thus, in a preferred aspect, a chemical reaction strategy forthe coupling step generally includes the formation of a phosphodiesterinternucleoside linkage. In accordance with this aspect, method a) andb) above are preferred.

In another aspect, when ligases are used for the ligation, suitableexamples include Taq DNA ligase, T4 DNA ligase, T7 DNA ligase, and E.coli DNA ligase. The choice of the ligase depends to a certain degree onthe design of the ends to be joined together. Thus, if the ends areblunt, T4 DNA ligase may be preferred, while a Taq DNA ligase may bepreferred for a sticky end ligation, i.e. a ligation in which anoverhang on each end is a complement to each other.

In a certain aspect of the invention enzymatic encoding is preferredbecause of the specificity enzymes provide. FIG. 17 discloses a varietyof methods for enzymatically encoding two or more reactants in thecoding part of the bifunctional molecule. The choice of encoding methoddepends on a variety of factors, such as the need for free reactants,the need for proximity, and the need for convenience. The enzymaticdouble encoding methods shown on FIG. 17 may easily be expanded totriple, quarto, etc. encoding.

In accordance with a certain embodiment functional entities are attachedto identifying tags, and each functional entity carries one or morereactive groups. All the functional entities react with each other togenerate the final product containing as many tags as functionalentities. The tags may be combined into a single coding part, usually anoligonucleotide through an intermolecular reaction or associationfollowed by cleavage of two of the linkers, as shown below:

Bold lines represent tags. Thin lines represent linkers or bonds. “*”denotes a priming site. In some aspects of the invention X is regardedas the chemical reactive site.

In one aspect of the above embodiment the tags are of oligonucleotides,which combine through chemical ligation or enzyme catalysed ligation.

Alternatively, the tags are coupled prior to the reaction of thefunctional entities. In that process the functional entities will becleaved from their tags or cleaved afterwards. E.g.

An embodiment of the above schematic representation comprises, when thetags are nucleotides, the combination of tags through chemical ligationor enzyme catalysed ligation.

Example 9 illustrates a multi component reaction in which tripleencoding is used. Thus after the reaction of three free reactants with achemical reactive site, the coding part is provided with threeidentifying tags by enzymatic ligation.

Building Blocks Capable of Transferring Functional Entities.

The following sections describe the formation and use of exemplarybuilding blocks capable of transferring a functional entity to areactive group of a bifunctional complex. A bold line indicates anoligonucleotide.

A. Acylation Reactions

General Route to the Formation of Acylating Building Blocks and the Useof these:

N-hydroxymaleimide (1) may be acylated by the use of an acylchloridee.g. acetylchloride or alternatively acylated in e.g. THF by the use ofdicyclohexylcarbodiimide or diisopropylcarbodiimide and acid e.g. aceticacid. The intermediate may be subjected to Michael addition by the useof excess 1,3-propanedithiol, followed by reaction with either4,4′-dipyridyl disulfide or 2,2′-dipyridyl disulfide. This intermediate(3) may then be loaded onto an oligonucleotide carrying a thiol handleto generate the building block (4). Obviously, the intermediate (2) canbe attached to the oligonucleotide using another linkage than thedisulfide linkage, such as an amide linkage and the N-hydroxymaleimidecan be distanced from the oligonucleotide using a variety of spacers.

The building block (4) may be reacted with an identifier oligonucleotidecomprising a recipient amine group e.g. by following the procedure: Thebuilding block (4) (1 nmol) is mixed with an amino-oligonucleotide (1nmol) in hepes-buffer (20 μL of a 100 mM hepes and 1 M NaCl solution,pH=7.5) and water (39 uL). The oligonucleotides are annealed together byheating to 50° C. and cooling (2° C./second) to 30° C. The mixture isthen left o/n at a fluctuating temperature (10° C. for 1 second then 35°C. for 1 second), to yield the product (5).

In more general terms, the building blocks indicated below is capable oftransferring a chemical entity (CE) to a recipient nucleophilic group,typically an amine group. The bold lower horizontal line illustrates thebuilding block and the vertical line illustrates a spacer. The5-membered substituted N-hydroxysuccinimide (NHS) ring serves as anactivator, i.e. a labile bond is formed between the oxygen atomconnected to the NHS ring and the chemical entity. The labile bond maybe cleaved by a nucleophilic group, e.g. positioned on a scaffold

Another building block which may form an amide bond is

R may be absent or NO₂, CF₃, halogen, preferably Cl, Br, or I, and Z maybe S or O. This type of building block is disclosed in Danish patentapplication No. PA 2002 0951 and US provisional patent application filed20 Dec. 2002 with the title “A building block capable of transferring afunctional entity to a recipient reactive group”. The content of bothpatent application are incorporated herein in their entirety byreference.

A nucleophilic group can cleave the linkage between Z and the carbonylgroup thereby transferring the chemical entity —(C═O)—CE′ to saidnucleophilic group.

B. Alkylation

General Route to the Formation of Alkylating/Vinylating Building Blocksand Use of these:

Alkylating building blocks may have the following general structure:

R¹ and R² may be used to tune the reactivity of the sulphate to allowappropriate reactivity. Chloro and nitro substitution will increasereactivity. Alkyl groups will decrease reactivity. Ortho substituents tothe sulphate will due to steric reasons direct incoming nucleophiles toattack the R-group selectively and avoid attack on sulphur.

An example of the formation of an alkylating building block and thetransfer of a functional entity is depicted below:

3-Aminophenol (6) is treated with maleic anhydride, followed bytreatment with an acid e.g. H₂SO₄ or P₂O₅ and heated to yield themaleimide (7). The ring closure to the maleimide may also be achievedwhen an acid stable O-protection group is used by treatment with Ac₂O,with or without heating, followed by O-deprotection. Alternativelyreflux in Ac₂O, followed by O-deacetylation in hot water/dioxane toyield (7). Further treatment of (7) with SO₂Cl₂, with or withouttriethylamine or potassium carbonate in dichloromethane or a higherboiling solvent will yield the intermediate (8), which may be isolatedor directly further transformed into the aryl alkyl sulphate by thequench with the appropriate alcohol, in this case MeOH, whereby (9) willbe formed.

The organic moiety (9) may be connected to an oligonucleotide, asfollows: A thiol carrying oligonucleotide in buffer 50 mM MOPS or hepesor phosphate pH 7.5 is treated with a 1-100 mM solution and preferably7.5 mM solution of the organic building block (9) in DMSO oralternatively DMF, such that the DMSO/DMF concentration is 5-50%, andpreferably 10%. The mixture is left for 1-16 h and preferably 2-4 h at25° C. to give the alkylating agent in this case a methylating buildingblock (10).

The reaction of the alkylating building block (10) with an amine bearingnascent bifunctional complex may be conducted as follows: Thebifunctional complex (1 nmol) is mixed the building block (10) (1 nmol)in hepes-buffer (20 μL of a 100 mM hepes and 1 M NaCl solution, pH=7.5)and water (39 uL). The oligonucleotides are annealed to each other byheating to 50° C. and cooled (2° C./second) to 30° C. The mixture isthen left o/n at a fluctuating temperature (10° C. for 1 second then 35°C. for 1 second), to yield the methylamine reaction product (11).

In more general terms, a building block capable of transferring achemical entity to a receiving reactive group forming a single bond is

The receiving group may be a nucleophile, such as a group comprising ahetero atom, thereby forming a single bond between the chemical entityand the hetero atom, or the receiving group may be an electronegativecarbon atom, thereby forming a C—C bond between the chemical entity andthe scaffold.

C. Vinylation Reactions

A vinylating building block may be prepared and used similarly asdescribed above for an alkylating building block. Although instead ofreacting the chlorosulphonate (8 above) with an alcohol, theintermediate chlorosulphate is isolated and treated with an enolate orO-trialkylsilylenolate with or without the presence of fluoride. E.g.

Formation of an Exemplary Vinylating Building Block (13):

The thiol carrying oligonucleotide in buffer 50 mM MOPS or hepes orphosphate pH 7.5 is treated with a 1-100 mM solution and preferably 7.5mM solution of the organic moiety (12) in DMSO or alternatively DMF,such that the DMSO/DMF concentration is 5-50%, and preferably 10%. Themixture is left for 1-16 h and preferably 2-4 h at 25° C. to give thevinylating building block (13).

The sulfonylenolate (13) may be used to react with amine carryingscaffold to give an enamine (14a and/or 14b) or e.g. react with acarbanion to yield (15a and/or 15b). E.g.

The reaction of the vinylating building block (13) and an amine ornitroalkyl carrying identifier may be conducted as follows:

The amino-oligonucleotide (1 nmol) or nitroalkyl-oligonucleotide (1nmol) identifier is mixed with the building block (1 nmol) (13) in 0.1 MTAPS, phosphate or hepes-buffer and 300 mM NaCl solution, pH=7.5-8.5 andpreferably pH=8.5. The oligonucleotides are annealed to the template byheating to 50° C. and cooled (2° C./second) to 30° C. The mixture isthen left o/n at a fluctuating temperature (10° C. for 1 second then 35°C. for 1 second), to yield reaction product (14a/b or 15a/b).Alternative to the alkyl and vinyl sulphates described above may equallyeffective be sulphonates as e.g. (31) (however with R″ instead as alkylor vinyl), described below, prepared from (28, with the phenyl groupsubstituted by an alkyl group) and (29), and be used as alkylating andvinylating agents.

Another building block capable of forming a double bond by the transferof a chemical entity to a recipient aldehyde group is shown below. Adouble bond between the carbon of the aldehyde and the chemical entityis formed by the reaction.

The above building block is comprised by the Danish patent applicationNo. DK PA 2002 01952 and the US provisional patent application filed 20Dec. 2002 with the title “A building block capable of transferring afunctional entity to a recipient reactive group forming a C═C doublebond”. The content of both patent applications are incorporated hereinin their entirety by reference.

D. Alkenylidation Reactions

General Route to the Formation of Wittig and HWE Building Blocks and Useof these:

Commercially available compound (16) may be transformed into the NHSester (17) by standard means, i.e. DCC or DIC couplings. An aminecarrying oligonucleotide in buffer 50 mM MOPS or hepes or phosphate pH7.5 is treated with a 1-100 mM solution and preferably 7.5 mM solutionof the organic compound in DMSO or alternatively DMF, such that theDMSO/DMF concentration is 5-50%, and preferably 10%. The mixture is leftfor 1-16 h and preferably 2-4 h at 25° C. to give the phosphine boundprecursor building block (18). This precursor building block is furthertransformed by addition of the appropriate alkylhalide, e.g.N,N-dimethyl-2-iodoacetamide as a 1-100 mM and preferably 7.5 mMsolution in DMSO or DMF such that the DMSO/DMF concentration is 5-50%,and preferably 10%. The mixture is left for 1-16 h and preferably 2-4 hat 25° C. to give the building block (19). As an alternative to this,the organic compound (17) may be P-alkylated with an alkylhalide andthen be coupled onto an amine carrying oligonucleotide to yield (19).

An aldehyde carrying identifier (20), may be formed by the reactionbetween the NHS ester of 4-formylbenzoic acid and an amine carryingoligonucleotide, using conditions similar to those described above. Theidentifier (20) reacts with (19) under slightly alkaline conditions toyield the alkene (21).

The reaction of monomer building blocks (19) and identifier (20) may beconducted as follows: The identifier (20) (1 nmol) is mixed withbuilding block (19) (1 nmol) in 0.1 M TAPS, phosphate or hepes-bufferand 1 M NaCl solution, pH=7.5-8.5 and preferably pH=8.0. The reactionmixture is left at 35-65° C. preferably 58° C. over night to yieldreaction product (21).

As an alternative to (17), phosphonates (24) may be used instead. Theymay be prepared by the reaction between diethylchlorophosphite (22) andthe appropriate carboxy carrying alcohol. The carboxylic acid is thentransformed into the NHS ester (24) and the process and alternativesdescribed above may be applied. Although instead of a simpleP-alkylation, the phosphite may undergo Arbuzov's reaction and generatethe phosphonate. Building block (25) benefits from the fact that it ismore reactive than its phosphonium counterpart (19).

E. Transition Metal Catalyzed Arylation, Hetaylation and VinylationReactions

Electrophilic building blocks (31) capable of transferring an aryl,hetaryl or vinyl functionality may be prepared from organic compounds(28) and (29) by the use of coupling procedures for maleimidederivatives to SH-carrying oligonucleotides described above.Alternatively to the maleimide the NHS-ester derivatives may be preparedfrom e.g. carboxybenzenesulfonic acid derivatives, be used by couplingof these to an amine carrying oligonucleotide. The R-group of (28) and(29) is used to tune the reactivity of the sulphonate to yield theappropriate reactivity.

The transition metal catalyzed cross coupling may be conducted asfollows: A premix of 1.4 mM Na₂PdCl₄ and 2.8 mM P(p-SO₃C₆H₄)₃ in waterleft for 15 min was added to a mixture of the identifier (30) andbuilding block (31) (both 1 nmol) in 0.5 M NaOAc buffer at pH=5 and 75mM NaCl (final [Pd]=0.3 mM). The mixture is then left o/n at 35-65° C.preferably 58° C., to yield reaction product (32).

Corresponding nucleophilic monomer building blocks capable oftransferring an aryl, hetaryl or vinyl functionality may be preparedfrom organic compounds of the type (35).

This is available by esterification of a boronic acid by a diol e.g.(33), followed by transformation into the NHS-ester derivative. TheNHS-ester derivative may then be coupled to an oligonucleotide, by useof coupling procedures for NHS-ester derivatives to amine carryingoligonucleotides described above, to generate building block type (37).Alternatively, maleimide derivatives may be prepared as described aboveand loaded onto SH-carrying oligonucleotides.

The transition metal catalyzed cross coupling is conducted as follows: Apremix of 1.4 mM Na₂PdCl₄ and 2.8 mM P(p-SO₃C₆H₄)₃ in water left for 15min was added to a mixture of the identifier (36) and the building block(37) (both 1 nmol) in 0.5 M NaOAc buffer at pH=5 and 75 mM NaCl (final[Pd]=0.3 mM). The mixture is then left o/n at 35-65° C. preferably 58°C., to yield template bound (38).

F. Reactions of Enamine and Enolether Monomer Building Blocks

Building blocks loaded with enamines and enolethers may be prepared asfollows: For Z═NHR (R=H, alkyl, aryl, hetaryl), a 2-mercaptoethylaminemay be reacted with a dipyridyl disulfide to generate the activateddisulfide (40), which may then be condensed to a ketone or an aldehydeunder dehydrating conditions to yield the enamine (41). For Z═OH,2-mercaptoethanol is reacted with a dipyridyl disulfide, followed byO-tosylation (Z═OTs). The tosylate (40) may then be reacted directlywith an enolate or in the presence of fluoride with aO-trialkylsilylenolate to generate the enolate (41). The enamine orenolate (41) may then be coupled onto an SH-carrying oligonucleotide asdescribed above to give the building block (42).

The building block (42) may be reacted with a carbonyl carryingidentifier oligonucleotide like (44) or alternatively an alkylhalidecarrying oligonucleotide like (43) as follows: The building block (42)(1 nmol) is mixed with the identifier (43) (1 nmol) in 50 mM MOPS,phosphate or hepes-buffer buffer and 250 mM NaCl solution, pH=7.5-8.5and preferably pH=7.5. The reaction mixture is left at 35-65° C.preferably 58° C. over night or alternatively at a fluctuatingtemperature (10° C. for 1 second then 35° C. for 1 second) to yieldreaction product (46), where Z═O or NR. For compounds where Z═NRslightly acidic conditions may be applied to yield product (46) withZ═O.

The building block (42) (1 nmol) is mixed with the identifier (44) (1nmol) in 0.1 M TAPS, phosphate or hepes-buffer buffer and 300 mM NaClsolution, pH=7.5-8.5 and preferably pH=8.0. The reaction mixture is leftat 35-65° C. preferably 58° C. over night or alternatively at afluctuating temperature (10° C. for 1 second then 35° C. for 1 second)to yield reaction product (45), where Z═O or NR. For compounds whereZ═NR slightly acidic conditions may be applied to yield product (45)with Z═O.

Enolethers type (13) may undergo cycloaddition with or withoutcatalysis. Similarly, dienolethers may be prepared and used, e.g. byreaction of (8) with the enolate or trialkylsilylenolate (in thepresence of fluoride) of an α,β-unsaturated ketone or aldehyde togenerate (47), which may be loaded onto an SH-carrying oligonucleotide,to yield monomer building block (48).

The diene (49), the ene (50) and the 1,3-dipole (51) may be formed bysimple reaction between an amino carrying oligonucleotide and theNHS-ester of the corresponding organic compound. Reaction of (13) oralternatively (31, R″=vinyl) with dienes as e.g. (49) to yield (52) ore.g. 1,3-dipoles (51) to yield (53) and reaction of (48) or (31,R″=dienyl) with enes as e.g. (50) to yield (54) may be conducted asfollows:

The building block (13) or (48) (1 nmol) is mixed with the identifier(49) or (50) or (51) (1 nmol) in 50 mM MOPS, phosphate or hepes-bufferbuffer and 2.8 M NaCl solution, pH=7.5-8.5 and preferably pH=7.5. Thereaction mixture is left at 35-65° C. preferably 58° C. over night oralternatively at a fluctuating temperature (10° C. for 1 second then 35°C. for 1 second) to yield template bound (52), (53) or (54),respectively.

Cross-Link Cleavage Building Blocks

It may be advantageous to split the transfer of a chemical entity to arecipient reactive group into two separate steps, namely a cross-linkingstep and a cleavage step because each step can be optimized. A suitablebuilding block for this two step process is illustrated below:

Initially, a reactive group appearing on the functional entity precursor(abbreviated FEP) reacts with a recipient reactive group, e.g. areactive group appearing on a scaffold, thereby forming a cross-link.Subsequently, a cleavage is performed, usually by adding an aqueousoxidising agent such as I₂, Br₂, Cl₂, H⁺, or a Lewis acid. The cleavageresults in a transfer of the group HZ-FEP- to the recipient moiety, suchas a scaffold.

In the above formula

-   -   Z is O, S, NR⁴    -   Q is N, CR¹    -   P is a valence bond, O, S, NR⁴, or a group C₅₋₇arylene,        C₁₋₆alkylene, C₁₋₆O-alkylene, C₁₋₆S-alkylene, NR¹-alkylene,        C₁₋₆alkylene-O, C₁₋₆alkylene-S option said group being        substituted with 0-3 R⁴, 0-3 R⁵ and 0-3 R⁹ or C₁-C₃ alkylene-NR⁴        ₂, C₁-C₃ alkylene-NR⁴C(O)R⁸, C₁-C₃ alkylene-NR⁴C(O)OR⁸, C₁-C₂        alkylene-O—NR⁴ ₂, C₁-C₂ alkylene-O—NR⁴C(O)R⁸, C₁-C₂        alkylene-O—NR⁴C(O)OR⁸ substituted with 0-3 R⁹,    -   B is a group comprising D-E-F, in which    -   D is a valence bond or a group C₁₋₆alkylene, C₁₋₆alkenylene,        C₁₋₆alkynylene, C₅₋₇arylene, or C₅₋₇heteroarylene, said group        optionally being substituted with 1 to 4 group R¹¹,    -   E is, when present, a valence bond, O, S, NR⁴, or a group        C₁₋₆alkylene, C₁₋₆alkenylene, C₁₋₆alkynylene, C₅₋₇arylene, or        C₅₋₇heteroarylene, said group optionally being substituted with        1 to 4 group R¹¹,    -   F is, when present, a valence bond, O, S, or NR⁴,    -   A is a spacing group distancing the chemical structure from the        complementing element, which may be a nucleic acid,    -   R¹, R², and R³ are independent of each other selected among the        group consisting of H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, C₄-C₈ alkadienyl, C₃-C₇ cycloalkyl, C₃-C₇        cycloheteroalkyl, aryl, and heteroaryl, said group being        substituted with 0-3 R⁴, 0-3 R⁵ and 0-3 R⁹ or C₁-C₃ alkylene-NR⁴        ₂, C₁-C₃ alkylene-NR⁴C(O)R⁸, C₁-C₃ alkylene-NR⁴C(O)OR⁸, C₁-C₂        alkylene-O—NR⁴ ₂, C₁-C₂ alkylene-O—NR⁴C(O)R⁸, C₁-C₂        alkylene-O—NR⁴C(O)OR⁸ substituted with 0-3 R⁹,    -   FEP is a group selected among the group consisting of H, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₄-C₈ alkadienyl, C₃-C₇        cycloalkyl, C₃-C₇ cycloheteroalkyl, aryl, and heteroaryl, said        group being substituted with 0-3 R⁴, 0-3 R⁵ and 0-3 R⁹ or C₁-C₃        alkylene-NR⁴ ₂, C₁-C₃ alkylene-NR⁴C(O)R⁸, C₁-C₃        alkylene-NR⁴C(O)OR⁸, C₁-C₂ alkylene-O—NR⁴ ₂, C₁-C₂        alkylene-O—NR⁴C(O)R⁸, C₁-C₂ alkylene-O—NR⁴C(O)OR⁸ substituted        with 0-3 R⁹,    -   where R⁴ is H or selected independently among the group        consisting of C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇        cycloalkyl, C₃-C₇ cycloheteroalkyl, aryl, heteroaryl, said group        being substituted with 0-3 R⁹ and    -   R⁵ is selected independently from —N₃, —CNO, —C(NOH)NH₂, —NHOH,        —NHNHR⁶, —C(O)R⁶, —SnR⁶ ₃, —B(OR⁶)₂, —P(O)(OR⁶)₂ or the group        consisting of C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₄-C₈ alkadienyl        said group being substituted with 0-2 R⁷,    -   where R⁶ is selected independently from H, C₁-C₆ alkyl, C₃-C₇        cycloalkyl, aryl or C₁-C₆ alkylene-aryl substituted with 0-5        halogen atoms selected from —F, —Cl, —Br, and —I;        and R⁷ is independently selected from —NO₂, —COOR⁶, —COR⁶, —CN,        —OSiR⁶ ₃, —OR⁶ and —NR⁶ ₂.        R⁸ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇        cycloalkyl, aryl or C₁-C₆ alkylene-aryl substituted with 0-3        substituents independently selected from —F, —Cl, —NO₂, —R³,        —OR³, —SiR³ ₃        R⁹ is ═O, —F, —Cl, —Br, —I, —CN, —NO₂, —OR⁶, —NR⁶ ₂,        —NR⁶—C(O)R⁸, —NR⁶—C(O)OR⁸, —SR⁶, —S(O)R⁶, —S(O)₂R⁶, —COOR⁶,        —C(O)NR⁶ ₂ and —S(O)₂NR⁶ ₂.

In a preferred embodiment Z is O or S, P is a valence bond, Q is CH, Bis CH₂, and R¹, R², and R³ is H. The bond between the carbonyl group andZ is cleavable with aqueous I₂.

Cleavable Linkers

A cleavable linker may be positioned between the target and a solidsupport or between the potential drug candidate and the identifierregion or any other position that may ensure a separation of the nucleicacid sequence comprising the codons from successful complexes fromnon-specific binding complexes. The cleavable linker may be selectivelycleavable, i.e. conditions may be selected that only cleave thatparticular linker.

The cleavable linkers may be selected from a large plethora of chemicalstructures. Examples of linkers includes, but are not limited to,linkers having an enzymatic cleavage site, linkers comprising a chemicaldegradable component, linkers cleavable by electromagnetic radiation.

Examples of Linkers Cleavable by Electromagnetic Radiation (Light)

O-Nitrobenzyl in Exo Position

For more details see Holmes C P. J. Org. Chem. 1997, 62, 2370-2380

For more details see Rajasekharan Pillai, V. N. Synthesis. 1980, 1-26

Dansyl Derivatives:

For more details see Rajasekharan Pillai, V. N. Synthesis. 1980, 1-26

Coumarin Derivatives

For more details see R. O, Schoenleber, B. Giese. Synlett 2003, 501-504

R¹ and R² can be either of the potential drug candidate and theidentifier, respectively.

Alternatively, R¹ and R² can be either of the target or a solid support,respectively.

R³═H or OCH₃

If X is O then the product will be a carboxylic acid

If X is NH the product will be a carboxamide

One specific example is the PC Spacer Phosphoramidite (Glen researchcatalog #10-4913-90) which can be introduced in an oligonucleotideduring synthesis and cleaved by subjecting the sample in water to UVlight (˜300-350 nm) for 30 seconds to 1 minute.

DMT=4,4′-Dimethoxytrityl

iPr=Isopropyl

CNEt=Cyanoethyl

The above PC spacer phosphoamidite is suitable incorporated in a libraryof complexes at a position between the indentifier and the potentialdrug candidate. The spacer may be cleaved according to the followingreaction.

R¹ and R² can be either of the encoded molecule and the identifyingmolecule, respectively. In a preferred aspect R² is an oligonucleotideidentifier and the R¹ is the potential drug candidate. When the linkeris cleaved a phosphate group is generated allowing for furtherbiological reactions. As an example, the phosphate group may bepositioned in the 5′end of an oligonucleotide allowing for an enzymaticligation process to take place.

Examples of Linkers Cleavable by Chemical Agents:

Ester linkers can be cleaved by nucleophilic attack using e.g. hydroxideions. In practice this can be accomplished by subjecting thetarget-ligand complex to a base for a short period.

R¹ and R² can be the either of be the potential drug candidate or theidentifier, respectively. R⁴⁻⁶ can be any of the following: H, CN, F,NO₂, SO₂NR₂.

Disulfide linkers can efficiently be cleaved/reduced byTris(2-carboxyethyl)phosphine (TCEP). TCEP selectively and completelyreduces even the most stable water-soluble alkyl disulfides over a widepH range. These reductions frequently required less than 5 minutes atroom temperature. TCEP is a non-volatile and odorless reductant andunlike most other reducing agents, it is resistant to air oxidation.Trialkylphosphines such as TCEP are stable in aqueous solution,selectively reduce disulfide bonds, and are essentially unreactivetoward other functional groups commonly found in proteins.

More details on the reduction of disulfide bonds can be found in Kirley,T. L. (1989), Reduction and fluorescent labeling ofcyst(e)ine-containing proteins for subsequent structural analysis, Anal.Biochem. 180, 231 and Levison, M. E., et al. (1969), Reduction ofbiological substances by water-soluble phosphines: Gamma-globulin.Experentia 25, 126-127.

Linkers Cleavable by Enzymes

The linker connecting the potential drug candidate with the identifieror the solid support and the target can include a peptide region thatallows a specific cleavage using a protease. This is a well-knownstrategy in molecular biology. Site-specific proteases and their cognatetarget amino acid sequences are often used to remove the fusion proteintags that facilitate enhanced expression, solubility, secretion orpurification of the fusion protein.

Various proteases can be used to accomplish a specific cleavage. Thespecificity is especially important when the cleavage site is presentedtogether with other sequences such as for example the fusion proteins.Various conditions have been optimized in order to enhance the cleavageefficiency and control the specificity. These conditions are availableand know in the art.

Enterokinase is one example of an enzyme (serine protease) that cut aspecific amino acid sequence. Enterokinase recognition site isAsp-Asp-Asp-Asp-Lys (DDDDK) (SEQ ID No: 1), and it cleaves C-terminallyof Lys. Purified recombinant Enterokinase is commercially available andis highly active over wide ranges in pH (pH 4.5-9.5) and temperature(4-45° C.).

The nuclear inclusion protease from tobacco etch virus (TEV) is anothercommercially available and well-characterized proteases that can be usedto cut at a specific amino acid sequence. TEV protease cleaves thesequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser (ENLYFQG/S) (SEQ ID NO:2)between Gln-Gly or Gln-Ser with high specificity.

Another well-known protease is thrombin that specifically cleaves thesequence Leu-Val-Pro-Arg-Gly-Ser (LVPAGS) (SEQ ID NO: 3) betweenArg-Gly. Thrombin has also been used for cleavage of recombinant fusionproteins. Other sequences can also be used for thrombin cleavage; thesesequences are more or less specific and more or less efficiently cleavedby thrombin. Thrombin is a highly active protease and various reactionconditions are known to the public.

Activated coagulation factor FX (FXa) is also known to be a specific anduseful protease. This enzyme cleaves C-terminal of Arg at the sequenceIle-Glu-Gly-Arg (IEGR) (SEQ ID NO: 4). FXa is frequently used to cutbetween fusion proteins when producing proteins with recombinanttechnology. Other recognition sequences can also be used for FXa.

Other types of proteolytic enzymes can also be used that recognizespecific amino acid sequences. In addition, proteolytic enzymes thatcleave amino acid sequences in an un-specific manner can also be used ifonly the linker contains an amino acid sequence in the complex molecule.

Other type of molecules such as ribozymes, catalytically activeantibodies, or lipases can also be used. The only prerequisite is thatthe catalytically active molecule can cleave the specific structure usedas the linker, or as a part of the linker, that connects the encodingregion and the displayed molecule or, in the alternative the solidsupport and the target.

A variety of endonucleases are available that recognize and cleave adouble stranded nucleic acid having a specific sequence of nucleotides.The endonuclease Eco RI is an example of a nuclease that efficientlycuts a nucleotide sequence linker comprising the sequence GAATTC alsowhen this sequence is close to the nucleotide sequence length. Purifiedrecombinant Eco RI is commercially available and is highly active in arange of buffer conditions. As an example the Eco RI is working invarious protocols as indicted below (NEBuffer is available from NewEngland Biolabs):

NEBuffer 1: [10 mM Bis Tris Propane-HCl, 10 mM MgCl₂, 1 mMdithiothreitol (pH 7.0 at 25° C.)],NEBuffer 2: [50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mMdithiothreitol (pH 7.9 at 25° C.)],NEBuffer 3: [100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl₂, 1 mMdithiothreitol (pH 7.9 at 25° C.)],NEBuffer 4: [50 mM potassium acetate, 20 mM Tris-acetate, 10 mMmagnesium acetate, 1 mM dithiothreitol (pH 7.9 at 25° C.)].Extension buffer: mM KCl, 20 mM Tris-HCl (Ph 8.8 at 25° C.), 10 mM(NH₄)₂SO₄, 2 mM MgSO₄ and 0.1% Triton X-100, and 200 μM dNTPs.

Nucleotides

The nucleotides used in the present invention may be linked together ina sequence of nucleotides, i.e. an oligonucleotide. Each nucleotidemonomer is normally composed of two parts, namely a nucleobase moiety,and a backbone. The back bone may in some cases be subdivided into asugar moiety and an internucleoside linker.

The nucleobase moiety may be selected among naturally occurringnucleobases as well as non-naturally occurring nucleobases. Thus,“nucleobase” includes not only the known purine and pyrimidinehetero-cycles, but also heterocyclic analogues and tautomers thereof.Illustrative examples of nucleobases are adenine, guanine, thymine,cytosine, uracil, purine, xanthine, diaminopurine,8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine,N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diamino-purine, 5-methylcytosine,5-(C³-C⁶)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine,isoguanine, inosine and the “non-naturally occurring” nucleobasesdescribed in Benner et al., U.S. Pat. No. 5,432,272. The term“nucleobase” is intended to cover these examples as well as analoguesand tautomers thereof. Especially interesting nucleobases are adenine,guanine, thymine, cytosine, 5-methylcytosine, and uracil, which areconsidered as the naturally occurring nucleobases.

Examples of Suitable Specific Pairs of Nucleobases are Shown Below:Natural Base Pairs

Synthetic Base Pairs

Synthetic Purine Bases Pairing with Natural Pyrimidines

Suitable Examples of Backbone Units are Shown Below (B Denotes aNucleobase):

The sugar moiety of the backbone is suitably a pentose but may be theappropriate part of an PNA or a six-member ring. Suitable examples ofpossible pentoses include ribose, 2′-deoxyribose, 2′-O-methyl-ribose,2′-flour-ribose, and 2′-4′-O-methylene-ribose (LNA). Suitably thenucleobase is attached to the 1′ position of the pentose entity.

An internucleoside linker connects the 3′ end of preceding monomer to a5′ end of a succeeding monomer when the sugar moiety of the backbone isa pentose, like ribose or 2-deoxyribose. The internucleoside linkage maybe the natural occurring phosphodiester linkage or a derivative thereof.Examples of such derivatives include phosphorothioate,methylphosphonate, phosphoramidate, phosphotriester, andphosphodithioate. Furthermore, the internucleoside linker can be any ofa number of non-phosphorous-containing linkers known in the art.

Preferred nucleic acid monomers include naturally occurring nucleosidesforming part of the DNA as well as the RNA family connected throughphosphodiester linkages. The members of the DNA family includedeoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine. Themembers of the RNA family include adenosine, guanosine, uridine,cytidine, and inosine.

Selection

Once the library has been formed in accordance with the methodsdisclosed herein, one must screen the library for chemical compoundshaving predetermined desirable characteristics. Predetermined desirablecharacteristics can include binding to a target, catalytically changingthe target, chemically reacting with a target in a manner whichalters/modifies the target or the functional activity of the target, andcovalently attaching to the target as in a suicide inhibitor. Inaddition to libraries produced as disclosed herein above, librariesprepared in accordance with method A and B below, may be screenedaccording to the present invention.

A. Display molecules can be single compounds in their final “state”,which are tagged individually and separately. E.g. single compounds mayindividually be attached to a unique tag. Each unique tag holdsinformation on that specific compound, such as e.g. structure, molecularmass etc.

B. A display molecule can be a mixture of compounds, which may beconsidered to be in their final “state”. These display molecules arenormally tagged individually and separately, i.e. each single compoundin a mixture of compounds may be attached to the same tag. Another tagmay be used for another mixture of compounds. Each unique tag holdsinformation on that specific mixture, such as e.g. spatial position on aplate.

The target can be any compound of interest. The target can be a protein,peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, substrate, metabolite, transition stateanalog, cofactor, inhibitor, drug, dye, nutrient, growth factor, cell,tissue, etc. without limitation. Particularly preferred targets include,but are not limited to, angiotensin converting enzyme, renin,cyclooxygenase, 5-lipoxygenase, IIL-10 converting enzyme, cytokinereceptors, PDGF receptor, type II inosine monophosphate dehydrogenase,β-lactamases, and fungal cytochrome P-450. Targets can include, but arenot limited to, bradykinin, neutrophil elastase, the HIV proteins,including tat, rev, gag, int, RT, nucleocapsid etc., VEGF, bFGF, TGFβ,KGF, PDGF, thrombin, theophylline, caffeine, substance P, IgE, sPLA2,red blood cells, glioblastomas, fibrin clots, PBMCs, hCG, lectins,selectins, cytokines, ICP4, complement proteins, etc.

The upper limit for the strength of the stringency conditions is thedisintegration of the complex comprising the displayed molecule and theencoding region. Screening conditions are known to one of ordinary skillin the art.

Complexes having predetermined desirable characteristics can bepartitioned away from the rest of the library while still attached to anucleic acid identifier tag by various methods known to one of ordinaryskill in the art. In one embodiment of the invention the desirableproducts are partitioned away from the entire library without chemicaldegradation of the attached nucleic acid such that the identifiernucleic acids are amplifiable. The part of the identifier comprising thecodons may then be amplified, either still attached to the desirablechemical compound or after separation from the desirable chemicalcompound.

In a certain embodiment, the desirable display molecule acts on thetarget without any interaction between the coding sequences attached tothe desirable display compound and the target. In one embodiment, thedesirable chemical compounds bind to the target followed by a partitionof the complex from unbound products by a number of methods. The methodsinclude plastic binding, nitrocellulose filter binding, columnchromatography, filtration, affinity chromatography, centrifugation, andother well known methods for immobilizing targets.

Briefly, the library is subjected to the partitioning step, which mayinclude contact between the library and a column onto which the targetis bound. All identifier sequences which do not encode for a reactionproduct having an activity towards the target will pass through thecolumn. Additional undesirable chemical entities (e.g., entities whichcross-react with other targets) may be removed by counter-selectionmethods. Desirable complexes are bound to the column and can be elutedby changing the conditions of the column (e.g., salt, etc.) or theidentifier sequence associated with the desirable chemical compound canbe cleaved off and eluted directly.

In a certain embodiment, the basic steps involve mixing the library ofcomplexes with the immobilized target of interest. The target can beattached to a column matrix or microtitre wells with directimmobilization or by means of antibody binding or other high-affinityinteractions. In another embodiment, the target and displayed moleculesinteract without immobilisation of the target. Displayed molecules thatbind to the target will be retained on this surface, while nonbindingdisplayed molecules will be removed during a single or a series of washsteps. The identifiers of complexes bound to the target can then beseparated by cleaving the physical connection to the synthetic molecule.It may be considered advantageously to perform a chromatography stepafter of instead of the washing step. After the cleavage of the physicallink between the synthetic molecule and the identifier, the identifiermay be recovered from the media and optionally amplified before thedecoding step.

In traditional elution protocols, false positives due to suboptimalbinding and washing conditions are difficult to circumvent and mayrequire elaborate adjustments of experimental conditions. However, anenrichment of more than 100 to 1000 is rarely obtained. The selectionprocess used in example 7 herein alleviates the problem with falsepositive being obtained because the non-specific binding complexes to alarge extent remain in the reaction chamber. The experiments reportedherein suggest that an enrichment of more than 107 can be obtained.

Additionally, chemical compounds which react with a target can beseparated from those products that do not react with the target. In oneexample, a chemical compound which covalently attaches to the target(such as a suicide inhibitor) can be washed under very stringentconditions. The resulting complex can then be treated with proteinase,DNAse or other suitable reagents to cleave a linker and liberate thenucleic acids which are associated with the desirable chemical compound.The liberated nucleic acids can be amplified.

In another example, the predetermined desirable characteristic of thedesirable product is the ability of the product to transfer a chemicalgroup (such as acyl transfer) to the target and thereby inactivate thetarget. One could have a product library where all of the products havea thioester chemical group, or similar activated chemical group. Uponcontact with the target, the desirable products will transfer thechemical group to the target concomitantly changing the desirableproduct from a thioester to a thiol.

Therefore, a partitioning method which would identify products that arenow thiols (rather than thioesters) will enable the selection of thedesirable products and amplification of the nucleic acid associatedtherewith.

There are other partitioning and screening processes which arecompatible with this invention that are known to one of ordinary skillin the art. In one embodiment, the products can be fractionated by anumber of common methods and then each fraction is then assayed foractivity. The fractionization methods can include size, pH,hydrophobicity, etc.

Inherent in the present method is the selection of chemical entities onthe basis of a desired function; this can be extended to the selectionof small molecules with a desired function and specificity. Specificitycan be required during the selection process by first extractingidentifiers sequences of chemical compounds which are capable ofinteracting with a non-desired “target” (negative selection, orcounter-selection), followed by positive selection with the desiredtarget. As an example, inhibitors of fungal cytochrome P-450 are knownto cross-react to some extent with mammalian cytochrome P-450 (resultingin serious side effects). Highly specific inhibitors of the fungalcytochrome could be selected from a library by first removing thoseproducts capable of interacting with the mammalian cytochrome, followedby retention of the remaining products which are capable of interactingwith the fungal cytochrome.

Enrichment

The present invention also relates to a method for determining theidentity of a chemical entity having a preselected property, comprisingthe steps of:

i) generating a tagged library of chemical entities by appending uniqueidentifier tags to chemical entities,ii) subjecting the library to a condition, wherein a chemical entity ora subset of chemical entities having a predetermined property ispartitioned from the remainder of the library,iii) recovering an anti-tag from the partitioned library, said anti-tagbeing capable of interacting with the unique identifier tag in aspecific manner, andiv) identifying the chemical entity/ies having a preselected function bydecoding the anti-tag.

The tag is appended the chemical entity by a suitable process. Notably,each chemical entity is appended a tag by a reaction involving achemical reaction between a reactive group of the chemical entity and areactive group of the tag, such as method A and B of the selectionsection. The attachment of the chemical entity may be directly orthrough a bridging molecule part. The molecule part may be any suitablechemical structure able to connect the chemical entity to the tag.

The anti-tag has the ability to interact with the unique identifier tagin a specific manner. The chemical structure of the anti-tag is to alarge extent dependant on the choice of unique tag. As an example, ifthe unique tag is chosen as an antibody, the anti-tag is selected as theepitope able to associate with the antibody. In general, it is preferredto use an anti-tag comprising a sequence of nucleotides complementary toa unique identifier tag.

The method may be performed without amplification in certainembodiments. However, when larger libraries are intended, it is ingeneral preferred to use an anti-tag which is amplifiable. Anti-tagscomprising a sequence of nucleotides may be amplified using standardtechniques like PCR. In the event the anti-tag is a protein, the proteinmay be amplified by attaching the mRNA which has encoded the synthesisthereof, generating the cDNA from the mRNA and subjecting said mRNA to atranslation system. Such system is described in WO 98/31700 the contentof which is incorporated herein by reference. An alternative method foramplifying a protein tag is to use phage-displayed proteins.

In the event the tag as well as the anti-tag is a sequence of nucleicacids, a tag:anti-tag hybrid may be formed prior to the subjecting thelibrary to partitioning conditions or subsequent to the partitioningstep. In some embodiments of the invention it is preferred to form thetag:anti-tag hybrid prior to the partition step in order to make theappended nucleotide sequence inert relative to the system as it is wellknown that certain sequences of nucleotides can bind to a target orcatalyse a chemical reaction.

The oligonucleotide anti-tag may be formed in a variety of ways. In oneembodiment of the invention, the anti-tag is formed as an enzymaticextension reaction. The extension comprises the initial annealing of aprimer to the unique identifier tag and subsequent extension of theprimer using a polymerase and dNTPs. Other types of extension reactionsmay also be contemplated. As an example ligases may be used to createthe primer starting from di- or trinucleotide substrates and theextension may be performed using a suitable polymerase.

It may be desirable to recover the anti-tag at various steps during theprocess. To this end it is preferred in some aspects of the invention toprovide the primer provided with a handle capable of binding to asuitable affinity partner. An arsenal of different handles and affinitypartners are available to the skilled person in the art. The most widelyused handle is biotin, which in general are also preferred according tothe present invention. Biotin binds to the affinity partner streptavidinor avidin. A standard technique in the laboratory is to recover abiochemical entity having attached a biotin using a solid phase coveredwith streptavidin. Suitably, the solid phase is a bead which may beseparated from the liquid after the binding action by rotation or amagnetic field in case the solid bead comprises magnetic particles.

In other aspects of the present invention, the anti-tag is provided as aseparate oligonucleotide. The separate oligonucleotide may be producedusing standard amidite synthesis strategies or may be provided usingother useful methods. It is in general preferred to provide theoligonucleotide by synthesis, at least in part, because the biotinamidite is easily incorporated in a nascent oligonucleotide strand.Following the addition of an oligonucleotide anti-tag to a liquidcomprising chemical entities tagged with complementing oligonucleotidetags a double stranded library is formed as a hybridisation productbetween the unique identifier tag and the anti-tag oligonucleotide.

As mentioned above, the anti-tag oligonucleotide may be provided with ahandle, such as biotin, capable of binding to an affinity partner, suchas streptavidin or avidin.

Following the addition of the anti-tag oligonucleotides to the taggedchemical entities, some of the oligonucleotides present in the media maynot find a partner. In one aspect of the invention it is preferred thatoligonucleotides not hybridised to a cognate unique identifier and/oranti-tag are transformed into a double helix. In other aspects of theinvention single stranded oligonucleotides are degraded prior to stepii) to avoid unintended interference.

The handle may be used to purify the library prior to or subsequent tothe partitioning step. In some embodiments of the invention, thepurification step is performed prior to the partitioning step to reducethe noise of the system. In another aspect the handle is used to purifythe partitioned library subsequent to step ii) in order to recover adouble stranded product which may be amplified.

The library is subjected to a condition in order to select chemicalentities having a property which is responsive to this condition. Thecondition may involve the exposure of the library to a target andpartitioning the chemical entities having an affinity towards thistarget. Another condition could be subjecting the library to a substrateand partitioning chemical entities having a catalytical activityrelative to this substrate.

The anti-tag can be formed subsequent to the partitioning step. In anaspect of the invention, the single stranded nucleotide serving as a tagis made double stranded while the chemical entity is attached to thetarget of an affinity partitioning. Optionally, in a repeatedtemperature cycle, a plurality of anti-tags may be formed as extensionproducts using the tag as template. In another aspect of the invention,the chemical entity bearing the single stranded oligonucleotide isdetached from the target and a complementing anti-tag is subsequentlyprepared.

In the event the anti-tag comprises a handle, this handle can be used topurify the partitioned library. The recovery of the anti-tag is thenperformed by melting off said anti-tag from a partitioned doublestranded library. Optionally, the amount of anti-tags may be multipliedby conventional amplification techniques, such as PCR.

The method according to the invention can be performed using a singlepartitioning step. Usually, it is preferred, however, to use more thanone partitioning step in order to select the candidate having thedesired properties from a large library. Thus, the recovered anti-tagsmay be mixed with the initial library or a subset thereof and the stepsof partitioning (step ii)) and recovery (step iii)) may is repeated adesired number of times. Optionally, single stranded moieties in themixture may be degraded or removed or made inert as described above.

Generally, the partitioned library obtained in step ii) is subjected toone or more further contacting steps using increasing stringencyconditions. The stringency conditions may be increased by increasing thetemperature, salt concentration, acidity, alkalinity, etc.

In one embodiment of the invention, the partitioned library is notsubjected to intermediate process steps prior to a repeated contactingstep. Especially, the partitioned library is not subjected tointermediate amplification of the anti-tag. This embodiment may be ofadvantage when relatively small libraries are used.

The method of the invention terminates with a decoding step, that is astep in which the identity of the chemical entity or entities aredeciphered by an analysis of the anti-tag. When the anti-tag is anoligonucleotide, the decoding step iv) may be performed by sequencing ananti-tag nucleotide. Various methods for sequencing are apparent for theskilled person, including the use of cloning and exposure to amicroarray.

The tags contain recognizing groups such as e.g. nucleotide sequence(s),epitope(s) a.o. The tags carries information of the entity to which itis attached, such as e.g. entity structure, mass, spatial position(plate information) etc. The tags may be composed of monoclonalantibodies, peptides, proteins, oligonucleotides, DNA, RNA, LNA, PNA,natural peptides, unnatural peptides, polymeric or oligomeric hydrazinoaryl and alkyl carboxylic acids, polymeric or oligomeric aminoxy aryland alkyl carboxylic acids, peptoids, other natural polymers oroligomers, unnatural polymers (molecular weight >1000 Da) or oligomers(molecular weight <1000 Da), small non-polymeric molecules (molecularweight <1000 Da) or large non-polymeric molecules (molecularweight >1000 Da).

In one preferred embodiment, entities consist of small non-polymericmolecules (molecular weight <1000 Da). Small molecules are generally thecompounds of interest in the quest for drug oral candidates. Especially,small molecules not occurring in Nature are of interest in the drugdiscovery process and in one aspect of the present invention the methodare designed to select a oral drug candidate. A variety of drugcandidate libraries are available on the market. The drug candidates ofthe library usually comprise a reactive group or a group which can bealtered into a reactive group. In one preferred aspect of the presentinvention each of the members of the drug candidate library is appendeda nucleic acid tag via said reactive group of the library member and areactive group on the nucleic acid. Preferably, the nucleic acid is anoligonucleotide.

In another aspect of the invention, entities consist of largenon-polymeric molecules (molecular weight >1000 Da). In still anotherembodiment, entities consist of polymeric molecules.

The tags and anti-tags may be composed of RNA linked to monoclonalantibodies, proteins, LNA, PNA, natural polypeptides, unnaturalpolypeptides, polymeric or oligomeric hydrazino aryl or alkyl carboxylicacids, polymeric or oligomeric aminoxy aryl or alkyl carboxylic acids,other natural polymers or oligomers, unnatural polymers (molecularweight >1000 Da) or oligomers (molecular weight <1000 Da), smallnon-polymeric molecules (molecular weight <1000 Da) or largenon-polymeric molecules (molecular weight >1000 Da).

Alternatively, anti-tags may be composed of DNA linked to monoclonalantibodies, proteins, LNA, PNA, natural polypeptides, unnaturalpolypeptides, polymeric or oligomeric hydrazino aryl or alkyl carboxylicacids, polymeric or oligomeric aminoxy aryl or alkyl carboxylic acids,other natural polymers or oligomers, unnatural polymers (molecularweight >1000 Da) or oligomers (molecular weight <1000 Da), smallnon-polymeric molecules (molecular weight <1000 Da) or largenon-polymeric molecules (molecular weight >1000 Da). Alternatively,anti-tags are just composed of oligonucleotides, DNA or RNA. In apreferred embodiment, anti-tags are composed of DNA. In anotherpreferred embodiment anti-tags are composed of RNA.

Anti-tags which are linked to DNA or RNA are also encoded by the DNA/RNAlinked to them, e.g. phage displayed or polysome displayed antibodies,peptides or proteins, and via DNA-templated synthesis of anti-tags,where the DNA encode the synthesis of the anti-tag, which is linked toits DNA during its synthesis.

Each chemical compound or group of compounds may be associated with atag through formation of a covalent or non-covalent bond. For covalentbond formation, tagging may involve, but is not limited to, theformation of a cycloaddition product, an alkylation product, anarylation product, an acylation product, an amide bond, a carboxylicester bond, a sulfonamide bond, a disulfide bond, an S-alkyl bond, anNR-alkyl bond, an O-alkyl bond, an aryl-vinyl bond, an alkyne-vinylbond, an oxime bond, an imine bond, a bicyclic product, a triazole, ahexene, a 7-Oxa-bicyclo[2.2.1]hept-2-ene derivative, a7-Aza-bicyclo[2.2.1]hept-2-ene derivative or a7-Methyl-7-aza-bicyclo[2.2.1]hept-2-ene. Non-covalent bonds may involve,but are not limited to, attachment via e.g. hydrogen bonding, van derWaals interactions, pi-stacking or through hybridization. Hybridizationmay be between complementary strands of DNA, RNA, PNA or LNA or mixturesthereof. In such case both the tag and the chemical compound carriessuch a strand complementary to each other. The tagged entity, compoundor mixture of compounds may be transformed into a new tagged entity,e.g. by transformation of the entity or by transformation of the tag.The transformation may be caused by either chemical or physicaltransformations such e.g. addition of reagents (e.g. oxidizing orreducing agents, pH adjustment a.o.) or subjection to UV-irradiation orheat.

The complex between tags and anti-tags may be formed on individuallytagged entities immediately after tagging. Alternatively, after mixingindividually tagged entities, either before or after the optionally useof library purification, or either before or after library enrichmentfor specific properties.

When tags and anti-tags are composed of nucleotides the complex consistsof a double stranded nucleotide, e.g. duplex DNA or hybrids DNA/RNA.

The purification handle (denoted “@”) may be connected to the anti-tag.The purification handle contains a recognizing group(s) such as e.g.nucleotide sequence(s), epitopes, reactive groups, high affine ligandsa.o. The purification handles may be composed of monoclonal antibodies,peptides, proteins, DNA, RNA, LNA, PNA, natural peptides, unnaturalpeptides, polymeric or oligomeric hydrazine aryl or alkyl carboxylicacids, polymeric or oligomeric aminoxy aryl or alkyl carboxylic acids,other natural polymers or oligomers, unnatural polymers (molecularweight >1000 Da) or oligomers (molecular weight <1000 Da), smallnon-polymeric molecules (molecular weight <1000 Da) or largenon-polymeric molecules (molecular weight >1000 Da). Purificationhandles may e.g. be a nucleotide sequence, biotin, streptavidin, avidin,“his-tags”, mercapto groups or disulfide/activated disulfide groups. Thepurification handle may be part of the anti-tag, e.g. in the case theanti-tag is nucleotide based or e.g. antibodies where part of theantibody may serve as epitope for another antibody (e.g. immobilizedantibody which serve as purification filter).

Purification filters contains components which associate, interact orreact with purification handles whereby a complex is formed. Thiscomplex allows separation of non-complexed tagged entities and complexedtagged entities. The purification filter contains a recognizing group(s)such as e.g. nucleotide sequence(s), epitopes, reactive groups, highaffine ligands a.o. The purification filter may be composed ofmonoclonal antibodies, peptides, proteins, DNA, RNA, LNA, PNA, naturalpeptides, unnatural peptides, polymeric or oligomeric hydrazino aryl oralkyl carboxylic acids, polymeric or oligomeric aminoxy aryl or alkylcarboxylic acids, other natural polymers or oligomers, unnaturalpolymers (molecular weight >1000 Da) or oligomers (molecular weight<1000 Da), small non-polymeric molecules (molecular weight <1000 Da) orlarge non-polymeric molecules (molecular weight >1000 Da). Purificationfilters may e.g. be a nucleotide sequence, biotin, strepdavidin, avidin,“his-tags”, mercapto groups or disulfide/activated disulfide groups.

The library is probed and enriched for properties. Properties may beaffinity, catalytic activity or membrane penetrating capability a.o.

Amplification may use PCR or RTPCR techniques. Anti-tags are amplifiablein some aspects of the invention. Anti-tags may be separated from tagsby use of physical or chemical means, such as e.g. UV-irradiation, heat,pH-adjustment, use of salt solutions a.o.

Isolated tagged entities may be identified either trough their tag oranti-tag. Identification may be accomplished by cloning of anti-tags andsequencing their DNA/RNA or through mass analysis of either taggedentities or anti-tags or complexes of anti-tags/tagged entities.

The library of tagged entities may involve 10-10²⁰ or 10-10¹⁴ or 10-10²or 10-10³ or 10²-10³ or 10²-10⁴ or 10³-10⁶ or 10³-10⁸ or 10³-10¹⁰ or10³-10¹⁴ or 10⁵-10⁶ or 10⁵-10⁸ or 10⁵-10¹⁰ or 10⁵-10¹⁴ or 10⁸-10¹⁴ or10¹⁴-10²⁰ entities.

Library complexes of tagged entities and anti-tags may be enriched forproperties prior to purification by use of purification handle andpurification filter or after purification.

The term unique, when used together with sequences of nucleotides,implies that at least one of the nucleobases and/or backbone entities ofthe sequence does not appear together with different chemical entities.Preferably, a specific sequence is unique due to fact that no otherchemical entities are associated with the same sequence of nucleobases.

Once the library has been formed, one must screen the library forchemical compounds having predetermined desirable characteristics.Predetermined desirable characteristics can include binding to a target,catalytically changing the target, chemically reacting with a target ina manner which alters/modifies the target or the functional activity ofthe target, and covalently attaching to the target as in a suicideinhibitor.

The target can be any compound of interest. The target can be a protein,peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, substrate, metabolite, transition stateanalog, cofactor, inhibitor, drug, dye, nutrient, growth factor, cell,tissue, etc. without limitation. Particularly preferred targets include,but are not limited to, angiotensin converting enzyme, renin,cyclooxygenase, 5-lipoxygenase, IIL-10 converting enzyme, cytokinereceptors, PDGF receptor, type II inosine monophosphate dehydrogenase,β-lactamases, and fungal cytochrome P-450. Targets can include, but arenot limited to, bradykinin, neutrophil elastase, the HIV proteins,including tat, rev, gag, int, RT, nucleocapsid etc., VEGF, bFGF, TGFβ,KGF, PDGF, thrombin, theophylline, caffeine, substance P, IgE, sPLA2,red blood cells, glioblastomas, fibrin clots, PBMCs, hCG, lectins,selectins, cytokines, ICP4, complement proteins, etc.

The stringency conditions under which the library are screened arenormally limited to such condition that maintain the hybridisationbetween the identifier tag and the anti-tag. High stringency conditionsmay be applied, however, followed by a renewed synthesis or attachmentof the anti-tag. Screening conditions are known to one of ordinary skillin the art.

Chemical compounds having predetermined desirable characteristics can bepartitioned away from the rest of the library while still attached to anucleic acid identifier tag by various methods known to one of ordinaryskill in the art. In one embodiment of the invention the desirableproducts are partitioned away from the entire library without chemicaldegradation of the attached nucleic acid such that the identifiernucleic acids are amplifiable. The identifier tag may then be amplified,either still attached to the desirable chemical compound or afterseparation from the desirable chemical compound.

In the most preferred embodiment, the desirable chemical compound actson the target without any interaction between the tag attached to thedesirable chemical compound and the target. In one embodiment, thedesirable chemical compounds bind to the target and the boundtag-desirable chemical compound-target complex can be partitioned fromunbound products by a number of methods. The methods includenitrocellulose filter binding, column chromatography, filtration,affinity chromatography, centrifugation, and other well known methods.

Briefly, the library is subjected to the partitioning step, which mayinclude contact between the library and a column onto which the targetis bound. All tags which have not formed hybridisation products with achemical entity-tag aggregate or those tags associated with undesirablechemical entities will pass through the column. Additional undesirablechemical entities (e.g., entities which cross-react with other targets)may be removed by counter-selection methods. Desirable complexes arebound to the column and can be eluted by changing the conditions of thecolumn (e.g., salt, etc.) or the tag associated with the desirablechemical compound can be cleaved off and eluted directly.

Additionally, chemical compounds which react with a target can beseparated from those products that do not react with the target. In oneexample, a chemical compound which covalently attaches to the target(such as a suicide inhibitor) can be washed under very stringentconditions. The resulting complex can then be treated with proteinase,DNAse or other suitable reagents to cleave a linker and liberate thenucleic acids which are associated with the desirable chemical compound.The liberated nucleic acids can be amplified.

In another example, the predetermined desirable characteristic of thedesirable product is the ability of the product to transfer a chemicalgroup (such as acyl transfer) to the target and thereby inactivate thetarget. One could have a product library where all of the products havea thioester chemical group. Upon contact with the target, the desirableproducts will transfer the chemical group to the target concomitantlychanging the desirable product from a thioester to a thiol. Therefore, apartitioning method which would identify products that are now thiols(rather than thioesters) will enable the selection of the desirableproducts and amplification of the nucleic acid associated therewith.

There are other partitioning and screening processes which arecompatible with this invention that are known to one of ordinary skillin the art. In one embodiment, the products can be fractionated by anumber of common methods and then each fraction is then assayed foractivity. The fractionization methods can include size, pH,hydrophobicity, etc.

Inherent in the present method is the selection of chemical entities onthe basis of a desired function; this can be extended to the selectionof small molecules with a desired function and specificity. Specificitycan be required during the selection process by first extractingidentifier sequences of chemical compounds which are capable ofinteracting with a non-desired “target” (negative selection, orcounter-selection), followed by positive selection with the desiredtarget. As an example, inhibitors of fungal cytochrome P-450 are knownto cross-react to some extent with mammalian cytochrome P-450 (resultingin serious side effects). Highly specific inhibitors of the fungalcytochrome could be selected from a library by first removing thoseproducts capable of interacting with the mammalian cytochrome, followedby retention of the remaining products which are capable of interactingwith the fungal cytochrome.

Following the selection procedure, anti-tags are recovered. The recoverymay be performed by subjecting the selected complexes to stringencyconditions which will detach the anti-tag sequences from the identifiertag. In the event the tag and the anti-tag are nucleic acids, thestringency conditions may be increased by increasing the temperaturegradually until the two strands of the double helix are melted apart.Further copies of anti-tag sequences may be provided by extension of theidentifier sequences using a suitable primer and a polymerase. In thealternative, the recovered anti-tag sequence and/or the identifiersequence tag may be subjected to PCR to form a double stranded product.The strands comprising the sequence that complements at least a part ofa unique identifier sequence are subsequently isolated.

The selected chemical entity may be attached to the target during theextension or amplification or may be detached from the target. In oneaspect of the invention, it is preferred that the target is immobilisedand the chemical compound remain attached to the target during theextension or amplification, to allow for easy recovery of the extensionor amplification product by simple elution. In another aspect theselected chemical entities are separated from the unique identifiersequences, prior to, simultaneous with or subsequent to the recovery ofthe enrichment sequences.

In order to recover the desired anti-tag sequences, it may beappropriate to provide the native as well as the amplified, if present,anti-tag sequences with one part of a molecular affinity pair. The onepart of a molecular affinity pair is also referred to herein as ahandle. The anti-tags may then be recovered by using the other part ofthe molecular affinity pair attached to a solid phase, which is possibleto isolate. The essential property of the molecular affinity pair isthat the two parts are capable of interacting in order to assemble themolecular affinity pair. In the biotechnological field a variety ofinteracting molecular parts are known which can be used as the molecularaffinity pair. Examples include, but are not restricted toprotein-protein interactions, protein-polysaccharide interactions,RNA-protein interactions, DNA-DNA interactions, DNA-RNA interactions,RNA-RNA interactions, biotin-streptavidin interactions, enzyme-ligandinteractions, antibody-ligand interaction, protein-ligand interaction,etc.

A suitable molecular affinity pair is biotin-streptavidin. The anti-tagsequences can be provided with biotin, e.g. by using a primer attachedto a biotin moiety in the amplification or extension step and contactingthe biotin tagged anti-tag sequence with beads coated with streptavidin.

After the recovery of the anti-tag sequences, these are contacted withthe initial library or a fraction thereof and an enriched library isallowed to be formed by the hybridisation of the anti-tag sequences tothe cognate sequence of the unique identifier tag.

The method according to the invention may be repeated one or more times.In a second round of the method, the part of the single stranded librarynot recognized by an anti-tag sequence may be cleared from the reactionmedia or the remaining part of the single stranded library may remain inadmixture with the enrich library. In general, it is not necessary toseparate the remaining part of the single stranded library from themedia before the enriched double stranded library is subjected to asecond contact with the target because conditions for the preselectedfunction usually are more stringent than the first round, wherefore themembers of the single stranded library presumably will not bind to thetarget. However, to reduce the noise of the system, it may be useful atsome events to withdraw from the media the members of the singlestranded initial library not mated with an anti-tag sequence. If theanti-tag sequences are provided with one part of a molecular affinitypair, like biotin, the chemical compounds of interest can be extractedfrom the media by treatment with immobilized streptavidin, e.g beadscoated with streptavidin.

As mentioned above, the conditions for performing the second or furtherselection step is generally more stringent than in the first orpreceding step. The increasing stringency conditions in sequentialselection rounds provide for the formation of a sub-library of chemicalcompounds which is narrowed with respect to the number but enriched withrespect to the desired property.

In the present description with claims, the terms nucleic acid,oligonucleotide, oligo, and nucleotides are used frequently. The termsnucleotide, nucleotide monomer, or mononucleotides are used to denote acompound normally composed of two parts, namely a nucleobase moiety, anda backbone. The back bone may in some cases be subdivided into a sugarmoiety and an internucleoside linker. Mononucleotides may be linked toeach other to form a oligonucleotide. Usually, the mononucleotides arelinked through an internucleoside linkage. The term nucleic acid coversmononucleotides as well as oligonucleotides. Usually, however, the termdenotes an oligonucleotide having from 2 to 30 mononucleotides linkedtogether through internucleoside linkers.

Determining the Coding Part of the Bifunctional Complex

The coding part of the identifier sequence present in the isolatedbifunctional molecules or the separated identifier oligonucleotides isdetermined to identify the chemical entities that participated in theformation of the display molecule. The synthesis method of the displaymolecule may be established if information on the functional entities aswell as the point in time they have been incorporated in the displaymolecule can be deduced from the identifier oligonucleotide. It may besufficient to get information on the chemical structure of the variouschemical entities that have participated in the display molecule todeduce the full molecule due to structural constraints during theformation. As an example, the use of different kinds of attachmentchemistries may ensure that a chemical entity on a building block canonly be transferred to a single position on a scaffold. Another kind ofchemical constrains may be present due to steric hindrance on thescaffold molecule or the functional entity to be transferred. In generalhowever, it is preferred that information can be inferred from theidentifier sequence that enable the identification of each of thechemical entities that have participated in the formation of the encodedmolecule along with the point in time in the synthesis history thechemical entities have been incorporated in the (nascent) displaymolecule.

Although conventional DNA sequencing methods are readily available anduseful for this determination, the amount and quality of isolatedbifunctional molecule may require additional manipulations prior to asequencing reaction.

Where the amount is low, it is preferred to increase the amount of theidentifier sequence by polymerase chain reaction (PCR) using PCR primersdirected to primer binding sites present in the identifier sequence.

In addition, the quality of the isolated bifunctional molecule may besuch that multiple species of bifunctional molecules are co-isolated byvirtue of similar capacities for binding to the target. In cases wheremore than one species of bifunctional molecule are isolated, thedifferent isolated species must be separated prior to sequencing of theidentifier oligonucleotide.

Thus in one embodiment, the different identifier sequences of theisolated bifunctional complexes are cloned into separate sequencingvectors prior to determining their sequence by DNA sequencing methods.This is typically accomplished by amplifying all of the differentidentifier sequences by PCR as described herein, and then using a uniquerestriction endonuclease sites on the amplified product to directionallyclone the amplified fragments into sequencing vectors. The cloning andsequencing of the amplified fragments then is a routine procedure thatcan be carried out by any of a number of molecular biological methodsknown in the art.

Alternatively, the bifunctional complex or the PCR amplified identifiersequence can be analysed in a microarray. The array may be designed toanalyse the presence of a single codon or multiple codons in anidentifier sequence.

Synthesis of Nucleic Acids

Oligonucleotides can be synthesized by a variety of chemistries as iswell known. For synthesis of an oligonucleotide on a substrate in thedirection of 3′ to 5′, a free hydroxy terminus is required that can beconveniently blocked and deblocked as needed. A preferred hydroxyterminus blocking group is a dimexothytrityl ether (DMT). DMT blockedtermini are first deblocked, such as by treatment with 3% dichloroaceticacid in dichloromethane (DCM) as is well known for oligonucleotidesynthesis, to form a free hydroxy terminus.

Nucleotides in precursor form for addition to a free hydroxy terminus inthe direction of 3′ to 5′ require a phosphoramidate moiety having anaminodiisopropyl side chain at the 3′ terminus of a nucleotide. Inaddition, the free hydroxy of the phosphoramidate is blocked with acyanoethyl ester (OCNET), and the 5′ terminus is blocked with a DMTether. The addition of a 5′ DMT-, 3′ OCNET-blocked phosphoramidatenucleotide to a free hydroxyl requires tetrazole in acetonitrilefollowed by iodine oxidation and capping of unreacted hydroxyls withacetic anhydride, as is well known for oligonucleotide synthesis. Theresulting product contains an added nucleotide residue with a DMTblocked 5′ terminus, ready for deblocking and addition of a subsequentblocked nucleotide as before.

For synthesis of an oligonucleotide in the direction of 5′ to 3′, a freehydroxy terminus on the linker is required as before. However, theblocked nucleotide to be added has the blocking chemistries reversed onits 5′ and 3′ termini to facilitate addition in the oppositeorientation. A nucleotide with a free 3′ hydroxyl and 5′ DMT ether isfirst blocked at the 3′ hydroxy terminus by reaction with TBS-Cl inimidazole to form a TBS ester at the 3′ terminus. Then the DMT-blocked5′ terminus is deblocked with DCA in DCM as before to form a free 5′hydroxy terminus. The reagent(N,N-diisopropylamino)(cyanoethyl)phosphonamidic chloride having anaminodiisopropyl group and an OCNET ester is reacted in tetrahydrofuran(THF) with the 5′ deblocked nucleotide to form the aminodiisopropyl-,OCNET-blocked phosphonamidate group on the 5′ terminus. Thereafter the3′ TBS ester is removed with tetrabutylammonium fluoride (TBAF) in DCMto form a nucleotide with the phosphonamidate-blocked 5′ terminus and afree 3′ hydroxy terminus. Reaction in base with DMT-Cl adds a DMT etherblocking group to the 3′ hydroxy terminus.

The addition of the 3′ DMT-, 5′ OCNET-blocked phosphonamidatednucleotide to a linker substrate having a free hydroxy terminus thenproceeds using the previous tetrazole reaction, as is well known foroligonucleotide polymerization. The resulting product contains an addednucleotide residue with a DMT-blocked 3′ terminus, ready for deblockingwith DCA in DCM and the addition of a subsequent blocked nucleotide asbefore.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the components of the identifier and the building block

FIG. 2 shows the principle of encoding by extension

FIG. 3 shows the extension region of the building block

FIG. 4 shows the components of the identifier and the building blockwith internal codons

FIG. 5 shows the principle of encoding by extension with specificannealing

FIG. 6 shows the encoding of scaffolded and polymer molecules

FIG. 7 shows the encoding by extension using three-strand assemblyprinciple

FIG. 8 shows encoding by extension using three-strand assembly principlewith specific annealing

FIG. 9 shows the synthesis of three-strand identifier-displayedmolecules using a solid-phase approach.

FIG. 10 shows the sequential reaction/extension using platform assembly.

FIG. 11 discloses a general scheme for alternating parallel synthesis ofa combinatorial library.

FIG. 12 discloses an encoding method using ligational encoding and afree reactant.

FIG. 13 discloses a library generating method in which a reaction isfollowed be an encoding step.

FIG. 14 discloses a library generation method using polymerase encoding.

FIG. 15 discloses various embodiments for single encoding methods.

FIG. 16 discloses a double encoding method.

FIG. 17 discloses various double encoding methods.

FIG. 18 discloses encoding using an loop building block.

FIG. 19 discloses a method in which a flexible linker is used in thebuilding block.

FIG. 20 discloses a gel showing the result of an experiment according toexample 6.

FIG. 21 discloses a triple encoding method.

FIG. 22 shows the setup used in example 9.

FIG. 23 shows the split-and-mix structure used in example 9.

FIG. 24 discloses an embodiment of library enrichment, amplification andidentification.

FIG. 25 shows an embodiment in which anti-tag sequences not hybridisedto a identifier sequence are made double stranded and thus inert.

FIG. 26 shows an embodiment in which an enrichment step is before thepurification step.

FIG. 27 shows a general principle of library enrichment, amplification,and identification.

FIG. 28 shows a general principle of library enrichment, amplification,and identification omitting the intermediate amplification step betweensubsequent enrichment procedures.

FIG. 29 shows a general principle of library enrichment, amplification,and identification in which the initial single stranded library is madedouble stranded prior to enrichment.

FIG. 30 shows a general principle for library enrichment, in which theanti-tag is not formed until after the one and more enrichmentprocesses.

FIG. 31 shows two gels reported in example 13.

FIG. 32 shows the result of the experiment reported in Example 14.

FIG. 33 shows the result of the experiment reported in Example 14.

FIG. 34 is a mass spectrogram showing the observed mass (7244.93 Da) forthe sample of example 1.

FIG. 35 is a mass spectrogram showing the observed mass (8369.32 Da) forthe sample of example 2.

FIG. 36 is a mass spectrogram showing the observed mass (7323.45 Da) forthe first sample of example 3.

FIG. 37 is a mass spectrogram showing the observed mass (6398.04 Da) forthe second sample of example 3.

FIG. 38 is a mass spectrogram showing the observed mass (6411.96 Da) forthe third sample of example 3.

FIG. 39 is a mass spectrogram showing the observed mass (7922.53 Da) forthe first sample of Example 4.

FIG. 40 is a mass spectrogram showing the observed mass (7936.99 Da) forthe second sample of example 4.

FIG. 41 is a mass spectrogram showing the observed masses or thetemplate (15452.14 Da) and the extended primer (15328.92 Da) in thefirst experiment of example 5.

FIG. 42 is a mass spectrogram showing the observed mass for the extendedprimer (28058.14 Da) for the second experiment of example 5.

FIG. 43 is a flow chart for the production of one embodiment of alibrary of bifunctional complexes, as set forth in Example 7. DF: Drugfragment/functional entity; B: Biotin; SA: Streptavidin.

FIG. 44 shows the retention time of the complex of Example 8 on asize-exclusion column.

FIG. 45 is a mass spectrogram showing the observed mass (66716.11 Da)for the loaded oligo in Example 9, section 9.1.

FIG. 46 is a mass spectrogram showing the observed masses for thestarting benzaldehyde loaded L1 oligo (A) and the UGI product (B) inExample 9, section 9.2.

FIG. 47 is a mass spectrogram showing the observed masses for thestarting benzaldehyde loaded L1 oligo (A), dilcetopiperazine (B), UGIproduct (C) and amine product (H) in Example 9, section 9.3.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 discloses in panel A a hybridisation product between a nascentbifunctional complex and a building block. The nascent bifunctionalcomplex, for short the Identifier, comprises an attachment entityconnected to an oligonucleotide identifier region by a linker moiety.The attachment entity may be a single recipient reactive group havingbeen adapted to receive a functional entity or may be a scaffoldstructure comprising one or more recipient reactive groups. In panel Athe attachment entity is indicated as a scaffold having four reactivegroups capable of receiving functional entities.

The building block comprises a functional entity attached to anoligonucleotide which is sufficiently complementary to the identifierregion to allow for a hybridisation product to be formed. The functionalentity is able to be transferred to the attachment entity through achemical reaction. The complementing identifier region further comprisesa unique codon at the 3′ or 5′ end thereof. The unique codon identifiesthe functional entity in an unequivocal way.

Following the formation of the hybridisation product between theidentifier and the building block, the functional entity and the uniqueanti-codon are transferred to the identifier. In an aspect of theinvention, the linker connecting the functional entity and thecomplementing identifier region is cleaved simultaneously with thereaction with the attachment entity resulting in a transfer of thefunctional entity to the attachment entity. Prior to, simultaneouslywith or subsequent to the transfer, the transcription of the codonoccurs. The transcription is performed by an enzyme capable ofpolymerisation or oligomerisation of oligonucleotides using a templateoligonucleotide to form a complementary stand. Usually a polymerase,such as the Pfu polymerase is used together with suitable dNTPs, i.e. amixture of ATP, CTP, GTP, and TTP, to form the unique codon as anextension of the identifier strand using the unique anti-codon of thebuilding block as template.

FIG. 1, panel B illustrates a typical setup for a second transfer offunctional entity. The identifier has been provided with a firstfunctional entity and has been extended by a codon. Furthermore, thecodon also comprises a binding region as an extension of the codon. Thebinding region is usually a constant region transferred to theidentifier in the first transfer cycle by the first building block. Theidentifier forms a hybridisation product with a second building block.The second building block comprises a second functional entity connectedto an oligonucleotide sufficient complementary to the identifier regionof the identifier to allow for a hybridisation. A part of thecomplementing identifier region comprises a non-coding region and aregion complementing the binding region. The non-coding region opposesthe codon transferred in the first cycle and the complementing bindingregion is complementary to the binding region to allow for ahybridisation which is sufficiently strong for an enzyme to bind to thehelix. A second unique anti-codon is attached to the complementarybinding region and identifies the second functional entity. The secondcodon is transferred to the identifier using the second anti-codon astemplate in the same manner as described above for the first codon.

FIG. 2 illustrates four cycles of functional entity and codon transfer.In the first cycle, a hybridisation product is formed between theidentifier and building block. The hybridisation product ensures thatthe functional entity and the scaffold are brought into close spatialproximity, thus increasing the probability that a reaction will takeplace. The formation of a duplex between the two oligonucleotides alsoprovides a binding region for a polymerase. In the presence of apolymerase, a mixture of dNTPs and a suitable puffer such as an aqueoussolution containing 20 mM HEPES-KOH, 40 mM KCl and 8 mM MgCl₂ and a pHadjusted to 7.4, the unique anti-codon (UA₁) is transferred to theidentifier as a codon.

After the transfer of functional entity and codon, respectively, thespent building block is separated from the identifier by increasing thestringency. Usually, the stringency is increased by a increasing thetemperature, changing the pH or by increasing the ionic strength. Afterthe rupture of the duple helix structure, the identifier is recovered.In one aspect of the invention the identifier is immobilized to ease theseparation from the spent building block. In another aspect the spentbuilding block is degraded chemically or enzymatically. Following therecovery of the identifier a new cycle can be initiated by contactingthe identifier with a further building block.

The final product after four cycles of transfer is a bifunctionalcomplex, which comprises a reaction product at one end and an encodingregion at the other. The reaction product comprises constituents fromthe transferred functional entities and the initial scaffold. Theencoding region comprises a genetic code for which entities that havebeen transferred in which order. Thus, the synthetic history may bedecoded from the encoding region.

FIG. 3 shows examples of the design of the coding area. Panel A, depictsa detailed view of an example of a design according to FIG. 1, panel B.The unique codon transferred in a first cycle is opposed by a partlymis-matching region. To compensate for the decrease in affinity abinding region is following the codon. The binding region is opposed bya matching complementary binding region of the building block.

In FIG. 3, panel B the unique codon incorporated in a first cycle isopposed by a second building block having incorporated in thecomplementing identifier region a neutral binding region. The neutralbinding region is not capable of discriminating between varieties ofunique codons, but is able to show some kind of affinity towards theeach of the codons. Usually, the neutral binding region comprises one ormore universal bases and more preferred the neutral binding regioncomprises a sequence of universal bases opposing at least a part of thecodon region on the identifier.

FIG. 4 shows a hybridisation product between an identifier and abuilding block wherein the identifier has internal codons and thebuilding block has corresponding anti-codons. The identifier region andthe complementing identifier region can also contain specific uniquecodons and anti-codons, respectively.

The use of internal codons is of particular importance when severalrounds of selection are anticipated, especially when the encodedmolecule is formed from a PCR product of a previous round. The internalanti-codons in the building block may completely or partly match theidentifier sequence or may comprise one or more universal bases toprovide for affinity but not for specificity. The role of the internalunique codons is only to guide the annealing between the identifiermolecule and the building block molecule. The correct encoding is takencare of by the unique codons which are created in the extension process.These unique codons are passed on to the next generation of moleculesand used to decode the synthetic history of the displayed molecules.This system will not be totally dependent on an accurate encodingfunction by the internal unique codons in order to pass the correctgenotype to the next generation of identifier molecules.

In panel A the hybridisation product provides for a spatial proximitybetween the functional entity and the attachment entity, thus increasingthe probability that a reaction occurs. The unique codon templates thecodon on the identifier sequence by an enzymatic extension reaction. Inpanel B a binding region is introduced between each unique codingsequence to provide for affinity of the two strands to each other eventhough one or more mis-matching bases appear in the codon:non-codingdomain of a previously used codon.

FIG. 5 shows an embodiment useful when an amplification step is involvedbetween selections. Initially, a library of complexes is produced asdepicted in FIG. 2. The library of the complexes may be subjected to aselection process. The selection process may involve presenting thedisplay molecule on the complex to a target and subsequent selecting thedisplay molecules which shows a desired interaction with the target. Itmay be advantageously to use relatively mild conditions during theselection process, to obtain a sub-library. The sub-library may bedecoded to obtain information on the synthetic history for the entiresub-library. However, it is usually preferred to reduce the sub-libraryfurther before a decoding is performed.

The sub-library may be reduced by subjecting it to the target again anduse more stringent conditions. However, to obtain a higher number ofeach of the members of the sub-library before a second selection, it isgenerally preferred to amplify the complex. Thus, a primer which isloaded with a scaffold is initially annealed to a primer site at one endof the encoding region. Subsequently a transcript is formed. A reverseprimer is preferably present to obtain a duple stranded PCR producthaving a scaffold attached thereto.

This PCR is the basis for the generation of en amplification of thesub-library. The identifier sequence is segregated into a number ofinternal unique codons, abbreviated IUC in the drawing. The number ofthe IUCs corresponds to the number of functional entities participatingin the formation of the display molecule. The sequence of the IUCsexpresses the identity of the individual functional entities and theorder of the IUCs indicates the order of reaction of the functionalentities. Preferably, a primer region is presented adjacent to thesequence of IUCs to allow for a later amplification of the nucleic acidsequence.

The sub-library is contacted with a plurality of building blockscomprising a transferable functional entity and an internal uniqueanti-codon (IUA) complementary to at least one of the IUCs. Thecomplementing identifier region is provided with sufficientcomplementarity to provide for a hybridisation with the oligonucleotideidentifier region. In a preferred embodiment the IUCs not identifying afunctional entity to be transferred is opposed in the complementaryidentifier region with a neutral binding region. As mentioned above theneutral binding region may comprise universal bases, i.e. bases thathave the ability to be paired with two or more of the naturallyoccurring nucleobases. Adjacent to the region comprising specificbase-pairing sequences and non-specific base-pairing sequences, i.e. thecomplementary identifier region is a unique anticodon (UA). The UAcomprises the same information as the IUA of the complementingidentifier region, typically the UA and the IUA has the same sequence onnucleotides.

The transfer step and the reaction step are conducted in several cyclesas described above to form a bifunctional complex. In FIG. 5 four cyclesare performed, however, it will be appreciated that less than cycles,such as 3 or 2 cycles can be performed to produce a reaction productcomprising constituent from 3 or 2 functional entities respectively.Also more, than four cycles may be performed, such as 5 to 20 to form amore diverse library of display molecules. The complexes resulting formthe cycles are a reaction product between the functional entities andthe scaffold, and an oligonucleotide. The oligonucleotide can be dividedinto a guiding region, that is, the region that guided the annealing ofthe individual building blocks, and an encoding region, which comprisesthe unique codons which have been transferred from the building blocksto the identifier.

Using the above encoding method, allows for the amplification of moreand more focused sub-libraries to obtain a sufficient amount of materialto allow decoding.

The encoding method shown in FIG. 6 can create both monomer and polymerencoded molecules. Panel A: Complex reaction products can be createdusing an attachment entity which has reacted with multiple functionalentities. Panel B: Polymers can be created using one attachment entitywith one reactive group allowing attachment with a functional entityhaving at least two reactive groups.

FIG. 7 illustrates a three strand assembly procedure for the encoding byextension principle. A: The identifier and building block can beassembled on an assembly platform. This assembly platform contains aunique anticodon region and a unique anticodon where these two elementsare directly linked through their sequences. There may be a connectingregion linking the unique anticodon region together with thecomplementing identifier region. B: Describes all the components of theidentifier, building block and the assembly platform used in theconsecutive reaction, where the identifier also contain a unique codonand a binding region and the assembly platform also contains anon-coding region and a complementing binding region.

In FIG. 8 it is shown that internal codons can also be used for thethree-strand assembly principle. This will be useful when selection willbe performed in multiple rounds with intermediate amplification steps.

In FIG. 8A the identifier comprises an attachment entity whereas in FIG.8B it comprises FE₁. Also, in FIG. 8B the identifier comprises a uniquecodon ER₁ and a binding region, whereas in FIG. 8A it does not. Also, inFIG. 8B the assembly platform comprises a non-coding region and acomplementing binding region between the complementing identifier regionand the unique anticodon, whereas in FIG. 8A it does not.

FIG. 9 shows a solid-phase three-strand displayed-molecule synthesis.The assembly platform molecule is attached to a solid support to allowsequential attachment of building blocks to the attachment entity.Different libraries of assembly platform molecules, which is extendedwith suitable non-coding regions and complementing binding regions, canbe used in each step in separate vials. This will allow the use ofidentical building block and identifier molecules in each step.

FIG. 10 shows the sequential transfer/extension using the assemblyplatform principle. Each well contains a library of platform molecules.The platform molecule is extended with one unique anticodon in thesubsequent wells. A library of identifier and building block molecule isadded to the first well which allows specific annealing and transfer offunctional entities. The reaction mixture is the transferred to the nextwells which finally generates the identifier-displayed library.

FIG. 11 discloses a general scheme for alternating parallel synthesis ofcombinatorial libraries. In a first step a nascent bifunctional moleculeis provided. The nascent bifunctional molecule comprises as one part ofthe molecule a reactive group, which may appear on a chemical scaffold,and some times referred to herein as a chemical reactive site. Anotherpart of the bifunctional molecule comprises a priming site for additionof a tag. The priming site may be a 3′-OH group or a 5′-phosphate groupof a nucleotide in case the tag is a nucleotide. The chemical reactivesite and the priming site may optionally be spaced by a linking group.In the event that the linking group is resent it may be a nucleotide ora sequence of nucleotides. The spacing entity may further comprise ahydrophilic linker, such as a polyethylene or polypropylene, to distancethe chemical reactive site from the nucleotide. Also comprised in thelinking moiety may be a selective cleavable linker that allows theexperimenter to separate the display molecule from the coding part.

The nascent bifunctional molecule is divided into a plurality ofcompartments, usually wells of a microtiter plate or similar equipmentthat allow easy handling of multiple spatially separated containers.Each of the compartments is reacted with a specific small moleculefragment, also referred to herein as a reactant. Thus, in a firstcompartment, the nascent bifunctional molecule is reacted with a firstsmall molecule fragment (F₁), in a second compartment; the nascentbifunctional molecule is reacted with a second small molecule fragment(F₂), etc. The number of compartments may in principle be indefinite,however, for practical reasons; the number is usually between 5 and5000, such as 10 and 500. In each of the compartments the small moleculefragments may be identical or different as the case may be. In eachcompartment, one, two, or more reactants may participate in thereaction. After the reaction between the drug fragment and the nascentbifunctional molecule has occurred in each compartment, a tag is added,said tag identifying the small molecule fragment. In certain aspects ofthe invention, the tag is a nucleic acid. Thus, in the firstcompartment, a first nucleic acid tag (T₁) is added to the priming siteof the reaction product, in the second compartment, a second nucleicacid tag (T₂) is added to the priming site of the second reactionproduct, etc. Various methods for enzymatic encoding are contemplatedand discussed herein. Following the enzymatic addition of the tags ineach of the compartments, the contents of the compartments arecollected.

In a second round the mixture of bifunctional molecules is split intocompartments again. The number of compartments of the second round neednot be the same as the number of compartments in the first round. Ineach compartment the products of the previous round serves as thenascent bifunctional molecule. Thus, a reactive group appearing on thereaction product between the scaffold and the small molecule fragment ofthe first round is reacted with one or more small molecule fragments ofthe second round. Thus, in a first compartment, the mixed reactionproducts of the first round are reacted with a first small moleculefragment (F₁), in a second compartment, the mixed reaction products ofthe first round are reacted with a second small molecule fragment (F₂),etc. The small molecule fragments F₁, F₂, . . . F_(x) of the secondround may be identical or different from the small molecule fragmentsused in the first round.

After the reactions have been allowed to occur, a tag specifying thesmall molecule fragment is added. The tag added in the first roundusually comprises a priming site that can be used for addition of thetag in the second round so as to produce a linear identifier comprisingthe tags. In the first compartment, the reacted product is added a firsttag which identifies the reactant of the second round that has reactedwith the reactive reaction site of the nascent bifunctional molecule; ina second compartment, the product reacted with the second small moleculefragment of the second round is added the tag identifying said reactant,etc. Following the addition of the tags in each compartment, the contentof the compartments are mixed in a common pool. Thesplit-reaction-combining cycle can be repeated an appropriate number oftimes to obtain a library of bifunctional molecules comprising a displaymolecule part and a coding part. The library may be used in a selectionprocess disclosed elsewhere herein.

Above, the general principle for split-and-mix is disclosed, in whichthe reaction of the small molecule fragment and the chemical reactionsite occurs prior to the encoding step. Obviously, the events can occurin the reverse order or simultaneously.

FIG. 12 schematically shows a 96 well microtiter plate to the left. Ineach well or in a selected number of wells, the process to the rightoccurs. Initially, a bifunctional molecule is provided. The bifunctionalmolecule comprise a chemical reaction site (oval) attached to a codon(rectangle) through a linker (line). To the left of the codon a bindingregion is provided. Next, a codon oligonucleotide and a splintoligonucleotide are added. The codon oligonucleotide is provided with acodon and flanking binding regions. The splint is designed withsequences complementing the binding region of the nascent bifunctionalmolecule and a binding region of the codon oligonucleotide such that theends abut each other under hybridisation conditions. The nascentbifunctional complex, the splint and the codon oligonucleotide forms ahybridisation product under appropriate conditions. A ligase is added tocouple the codon oligo to the nascent bifunctional complex. In a secondstep, a drug fragment, i.e. a reactant, is added and conditionsproviding for a reaction with the chemical reaction site is instituted.

Then the content of each well is combined and, optionally, divided intoa range of wells again for a second round of reaction and encoding. Infinal step, the combined contents of the wells are used in a selectionor partition step, as disclosed herein.

FIG. 13 outlines an embodiment with the encoding and reaction stepreversed compared to the embodiment shown in FIG. 12. In a variety ofwells a nascent bifunctional complex having a reactive group (Rx)attached to an oligonucleotide (horizontal line) is dispensed. In afirst step, the reactive group in each compartment is reacted with areactant, in a second step a codon oligonucleotide and a splint is addedtogether with a ligase to ligate covalently the codon oligonucleotide tothe reacted nascent bifunctional complex, and in a third step theligation product is recovered. The content of the wells may subsequentlybe combined and used as a library of bifunctional complexes or recycledfor another round of reaction and addition of tag.

FIG. 14 discloses the use of the library produced in accordance FIG. 13,or any other library having a coding part and display molecule part, ina further round. Initially, the combined contents of the wells from theembodiment of FIG. 13 are dispensed in separate wells. Then ananti-codon oligonucleotide having a binding region which iscomplementary to the binding region of the nascent bifunctional moleculeis added under hybridisation conditions, i.e. conditions which favourthe assembly of the hybridisation product between the nascentbifunctional complex and the anti-codon oligonucleotide. Subsequently,or simultaneously with the addition of the anti-codon oligonucleotide, apolymerase, a collection of dNTP (usually, dATP, dGTP, dCTP, and dTTP),and appropriate salts and buffer are added to provide for an extensionto occur. The extension (dotted arrow) transcribe the anti-codon to theidentifier, thus attaching a tag that encodes the identity of thereactant subsequently reacted at the chemical reaction site. Theanti-codon oligonucleotide is connected to a biotin (B) to allow forremoval of the oligonucleotide.

FIG. 15 discloses a scheme of various encoding methods combined with acollection of reactants. All the combinations are in according theinvention.

Free Reactant/Polymerase Encoding:

A nascent bifunctional complex comprises a scaffold (=chemical reactionsite) comprising a reactive group and an oligonucleotide part comprisinga codon identifying the scaffold. The codon is associated with anoligonucleotide binding region capable of forming a hybridisationproduct with a complementing binding region of an anti-codonoligonucleotide. The hybridisation product is subjected to an extensionreaction, in which the scaffold oligonucleotide is extended over theanti-codon, thereby providing the scaffold oligonucleotide with a codon.Subsequent, simultaneously with or prior to the extension reaction, afree reactant coded for by the anti-codon is reacted with the scaffold.

Zipper Building Block/Polymerase:

A nascent bifunctional complex comprises a scaffold (=chemical reactionsite) comprising a reactive group and an oligonucleotide part comprisinga codon identifying the scaffold. The codon is associated with twooligonucleotide binding region capable of forming a hybridisationproduct with a complementing binding region of an anti-codonoligonucleotide and a complementing binding region of the reactant. Thehybridisation product is subjected to an extension reaction, in whichthe scaffold oligonucleotide is extended over the anti-codon, therebyproviding the scaffold oligonucleotide with a codon. Subsequent,simultaneously with or prior to the extension reaction, a functionalentity coded for by the anti-codon is reacted with the scaffold. Theselection of polymerase may determine the order of reaction and encodingas some polymerase, such as Sequenase, displaces the binding regionattached to the functional entity, while other polymerases, like Taqpolymerase, do not perform the displacement of the binding region. Whena zipper building block is used a close proximity between the scaffoldand the functional entity is obtained thereby promoting a reaction totake place.

E2 Building Block/Polymerase Encoding:

A nascent bifunctional complex comprises a chemical scaffold and anoligonucleotide part comprising the codon identifying the scaffold. Theoligonucleotide part comprises two binding region on each sides of thecodon. An E2 building block anneals to the scaffold oligonucleotide suchthat the functional entity comes in close proximity as to the scaffoldand a double helix is formed just before the anti-codon, thus enable apolymerase to recognize the double helix as a binding area. Applyingappropriate conditions and substrates enable the extension of theidentifier oligonucleotide over the anti-codon, thus transcribing thegenetic information of the function entity to the identifier. Opposingthe scaffold codon is a stretch of universal binding nucleotides, suchas inosine. Use of an E2 building block allows for one-pot synthesis ofa library.

Loop Building Block/Polymerase Encoding:

A nascent bifunctional complex comprises a chemical scaffold and anoligonucleotide part comprising the codon identifying the scaffold. Theoligonucleotide part comprises two binding region on each sides of thecodon. A loop building block anneals to the scaffold oligonucleotidesuch that the functional entity comes in close proximity as to thescaffold and a double helix is formed just before the anti-codon, thusenable a polymerase to recognize the double helix as a binding area.Applying appropriate conditions and substrates enable the extension ofthe identifier oligonucleotide over the anti-codon, thus transcribingthe genetic information of the function entity to the identifier. As nosequence on the building block complements the scaffold codon sequence,this codon sequence loops out. Use of a loop building block allows forone-pot synthesis of a library.

N Building Block/Polymerase Encoding:

A nascent bifunctional complex comprises a chemical scaffold attached toa scaffold codon through a linker. On one or each side of the codon abinding region is present. An N building block comprises a bindingregion which is complementary to the scaffold binding region and ananti-codon. A functional entity is attached to the codon or a bindingregion. Under hybridisation conditions the complementary binding regionshybridise and a polymerase extends in both directions, therebytransferring the genetic information of the anti-codon to theoligonucleotide covalently connected to the scaffold. Before, after orsimultaneously with the extension reaction, the reaction between thefunctional entity and the scaffold may take place. Usually, thefunctional entity is attached to the anti-codon oligonucleotide via acleavable linker so as to allow for transfer of the functional entity tothe scaffold structure.

Free Reactant/Ligase:

A scaffold entity is attached to an oligonucleotide comprising a codon.The scaffold oligonucleotide further comprises a priming site to which acodon oligonucleotide is ligated. The ligation is performed by a ligase.The ligation can take place in a single stranded or double strandedform. In the single stranded form, a 3′ OH (or 5′-phosphate) of thescaffold oligonucleotide is ligated to a 5′-phosphate (or 3′-OH) of thecodon oligonucleotide. In the double stranded form, an oligonucleotidecomplementing the ends of the scaffold and codon oligonucleotides,respectively, is used and designed so that the ends abuts each other.Optionally, the ligation occurs between two double strandedoligonucleotides, i.e. a double stranded scaffold oligonucleotide withan over hang (“sticky end”) is ligated to a double stranded codonoligonucleotide provided with a complementing overhang. The type ofligation depends on the selected enzyme. Usually, the double strandedligation is preferred because the reaction is faster due to the guidingeffect of the oligonucleotide complementing the ends. The complementingoligonucleotide is also referred to herein as the splintoligonucleotide. Following, preceding, or simultaneously with theligation of the codon oligonucleotide to the scaffold oligonucleotide areaction between the free reactant and the scaffold takes place.

Zipper Building Block/Ligase:

A scaffold entity is attached to an oligonucleotide comprising a codonand binding region between the scaffold and the codon. The scaffoldoligonucleotide further comprises a priming site to which a codonoligonucleotide is ligated. The ligation is performed by a ligase. Theligation can take place in a single stranded or double stranded form. Inthe single stranded form, a 3′ OH (or 5′-phosphate) of the scaffoldoligonucleotide is ligated to a 5′-phosphate (or 3′-OH) of the codonoligonucleotide. In the double stranded form, an oligonucleotidecomplementing the ends of the scaffold and codon oligonucleotides,respectively, is used and designed so that the ends abuts each other.Optionally, the ligation occurs between two double strandedoligonucleotides, i.e. a double stranded scaffold oligonucleotide withan over hang (“sticky end”) is ligated to a double stranded codonoligonucleotide provided with a complementing overhang. The type ofligation depends on the selected enzyme. Usually, the double strandedligation is preferred because the reaction is faster due to the guidingeffect of the oligonucleotide complementing the ends. The complementingoligonucleotide is also referred to herein as the splintoligonucleotide. A zipper building block is a functional entity attachedto a binding oligonucleotide. The binding oligonucleotide iscomplementing the binding region of the scaffold oligonucleotide, thusforming a hybridisation product under hybridisation conditions.Following, preceding, or simultaneously with the ligation of the codonoligonucleotide to the scaffold oligonucleotide a reaction between thefunctional entity and the scaffold takes place. The use of the bindingregion on the reactant ensures a close proximity between the functionalentity and the scaffold.

E2 Building Block/Ligational Encoding:

Initially is provided a nascent bifunctional complex comprising ascaffold attached to an oligonucleotide, said oligonucleotide comprisinga codon and a binding region between the scaffold codon and the scaffoldcodon. The scaffold oligonucleotide also comprises a priming site towhich a codon oligonucleotide can be ligated. The scaffoldoligonucleotide is hybridised to an E2 building block which carries adouble stranded part. The oligonucleotide complementing the anticodon asligated to the scaffold oligonucleotide using the E2 building block as atemplate. Before, after or simultaneously with the ligation a reactiontakes place between the functional entity and the scaffold.

Loop Building Block/Ligational Encoding:

A bifunctional complex is provided comprising a scaffold attached to anoligonucleotide, wherein the scaffold oligonucleotide comprises a codonflanked by two binding regions. A loop building block is provided whichhas binding regions complementing the binding regions of the scaffoldoligonucleotide. Upon hybridisation, the codon part of the scaffoldoligonucleotide loops out. The loop building block also comprises adouble stranded codon part. The oligonucleotide complementing theanti-codon part of the loop building block is ligated to the freebinding region of the scaffold oligonucleotide. Before, after orsimultaneously with the ligation a reaction takes place between thefunctional entity and the scaffold.

N Building Block/Ligational Encoding:

A nascent bifunctional complex is initially provided in which a scaffoldvia a suitable linker is attached the codon identifying said scaffold orattached to a binding region connect to the codon. A building blockhaving a functional entity connected to a codon is the ligated to thescaffold oligonucleotide to connect the scaffold oligonucleotide withfunctional entity oligonucleotide. The ligation may be performed in asingle stranded or in a double stranded state, depending on theparticular enzyme selected for the ligation. Subsequently, thefunctional entity is reacted with the scaffold. In the alternative, thefunctional entity and the scaffold are reacted prior to ligation of therespective oligonucleotides.

When a round, i.e. a reaction with and a tagging of the nascentbifunctional complex, has been completed in accordance with any of theabove encoding methods, a new round maybe in initialized according toany of the above reaction/encoding methods. Thus, the encoding andreaction in a first round may be the same or different in a subsequentsecond or further round. A single bifunctional complex or a library ofcomplexes may be generated. When a library is contemplated,one-pot-synthesis can be conducted with the building blocks in which acovalent link between the functional entity and the codon/anti-codon isused, i.e. the columns of E2 building block, loop building block, and Nbuilding block. Split and mix synthesis can be performed, when nocovalent link between the functional entity/reactant and thecodon/anti-codon is present, i.e. in the columns indicating the freereactant and the zipper building block.

FIG. 16 shows a double encoding method, i.e. a method for encoding twoor more reactants in one go. In certain embodiments, the multipleencoding methods allow for multi reaction between reactants andscaffold. Initially, a scaffold connected to an oligonucleotidecomprising a hybridisation region, a scaffold codon and a binding regionis annealed to an E2 building block. Subsequently, an extension isperformed in which the anti-codon of the building block is transferredto the identifier. Several polymerases form an overhang of one or moresingle stranded nucleotides. This overhang is used in the presentinvention to attach an anti-codon oligo and allow the polymerase tofurther extent the identifier oligonucleotide over the anti-codon regionof the anti-codon oligonucleotide. The transfer of the information ofthe anti-codon oligonucleotide allows for encoding a third free reactantC. The annealing between the oligonucleotide carrying A and theoligonucleotide carrying B provide for a close proximity between A and Band thus a high local concentration. Thus, when the free reactant C isadded a reaction between the three components is favoured. One advantageof double encoding is that it is possible to exchange solvent, such thatthe reaction not necessarily must take place in the same solvent as theextension occurs.

To the right is illustrated an example, in which the above method isapplied on 100 different scaffold oligonucleotides and 100 buildingblocks. The hybridisation product between the scaffold oligonucleotidesand the building block oligonucleotides is divided into 100 differentwells. In each of the wells the extension, addition of anti-codonoligonucleotide and reaction with specific free reactant is allowed. Intotal 10⁶ different bifunctional molecules are generated.

FIG. 17 discloses various methods for performing double encoding. In allthe examples, the encoding is shown to occur prior to reaction, but itwill be within the ambit of the skilled person to perform the reactionfirst and then the encoding. When a library is contemplated, it ispossible to conduct the reaction in a single container (one-potsynthesis) using the N building blocks in combination with any of theencoding methods. For the remaining reactants it is necessary to conductone or more split-and-mix step. In the combination of the zipperbuilding block, E2 building block, and the loop building block with anyof the encoding methods a single split-and-mix step is necessary,whereas two split-and-mix steps are necessary for the free reactant incombination with any encoding method. The scheme makes it possible forthe skilled person to select a reaction/encoding method which is usefulfor a specific reaction. If triple-, quadro-, or multi encoding iscontemplated, it is possible to perform such encoding using anembodiment of the double encoding scheme in combination with anembodiment of the single encoding scheme of FIG. 15 one or more times toarrive at an encoding/reaction method that suits the need for a specificchemical reaction.

FIG. 21 discloses a triple encoding method. Initially, a scaffoldattached to a scaffold oligonucleotide is provided. The scaffold isattached to a binding region the scaffold oligonucleotide, and thescaffold oligonucleotide is further provided with a codon. The twobuilding blocks of the E2 type is annealed to the scaffoldoligonucleotide, thereby bringing the functional entities BB1 and BB2into close proximity with the scaffold. Simultaneously, prior orsubsequent to the addition the building blocks a codon oligonucleotidecoding for a third reactant (BB3) is provided which comprises a partcomplementing a nucleotide sequence of the first building block. Thecomponents of the system are allowed to hybridise to each other and apolymerase and a ligase is provided. The polymerase performs anextension where possible and the ligase couples the extendedoligonucleotides together so as to form a double stranded product.Following the encoding process, the third reactant is added andconditions are provided which promote a reaction between the scaffoldand the reactants. Finally, a selection is used to select reactionproducts that perform a certain function towards a target. Theidentifying oligonucleotides of the selected bifunctional complexes areamplified by PCR and identified.

To the right a particular embodiment for carrying out the presentinvention is indicated. Accordingly, each codon is 5 nucleotides inlength and the binding regions flanking the scaffold are 20 nucleotideseach. The building blocks designed to hybridise to the binding regionsof the scaffold comprises a 20 nucleotide complementing sequence as wellas a 5 nucleotide codon.

An embodiment of the enrichment method of the present invention is shownon FIG. 24. Initially, each chemical entity (denoted by letters A, B, C,. . . ) in a library is attached to a unique identifier tag (denoted a,b, c, . . . ). The identifier tag comprises information about thatparticular compound or group of compounds with respect to e.g.structure, mass, composition, spatial position, etc. In a second step,tagged chemical compounds are combined with a set of anti-tag sequences(denoted a′, b′, c′, . . . ). Each anti-tag sequence carries a handle,like biotin, for purification purposes. The anti-tag sequences comprisea segment which is complementary to a sequence of the identifiersequence. The combination of anti-tag sequences and identifier sequencesare allowed to form hybridisation products. Optionally, there may betagged chemical entities present which have not been recognized by ananti-tag. In a third step, the sequences carrying a handle are removed,i.e. the tagged chemical compounds are left in the media while thematter comprising a handle is transferred to a second media. In theevent, the handle is biotin it may be transferred to a second mediausing immobilized streptavidin.

The purified matter may comprise anti-tag sequences not hybridised to acognate sequence. As these anti-tag sequences are not coupled to achemical compound to be selected for, the enrichment sequences mayremain in the media. However, in some applications it may be preferablyto make the excess anti-tag sequences double stranded, as illustrated inFIG. 25, because the double helix normally is inert relative to theselection procedure. The excess anti-tag sequences may be transformedinto the double helix state by the use of a primer together with asuitable polymerase and nucleotide triphosphates.

The purified fraction is in step 4 is subjected to a selection process.The selection comprises probing for a set of properties, e.g. but notlimited to affinity for a specific protein. In such a case, entitieswhich do not bind to the specific protein will be eliminated. Anti-tagscomplexed to entities binding to the specific protein may berecovered/be isolated through e.g. the use of its purification handle.

In step 5 isolated anti-tags are optionally amplified through the use ofPCR or RTPCR.

In step 6, the initial library of tagged entities produced in step 1,may undergo further rounds of complexation and screening, i.e. theanti-tags from step 5 may be added the library of tagged entities ofstep 1 and then be submitted to step 3, step 4 and step 5. Step 6 may berepeated.

In step 7, the isolated anti-tags of step 5 may be cloned and theiridentity be revealed. E.g. in the case of DNA, sequencing may be appliedwhereby the identity of specific entities with selected properties inthe library of tagged entities will be revealed.

The embodiment shown in FIG. 26 resembles that of FIG. 24 except thatthe non-complexed components are rendered inert, e.g. if the tags and/oranti-tags are composed of single stranded DNA or RNA, they may betransformed into double stranded DNA, RNA or a hybrid thereof. This maybe accomplished by use of a primer, nucleotide triphosphates and apolymerase or transcriptase. Furthermore, the sequence of purification(by use of the purification handle on anti-tags) and probing forproperties is changed compared to the method of FIG. 24.

In FIG. 27, step 1, a number of entities (denoted by letters A, B, C . .. ), being it mixtures or single compounds are attached to a unique tagmore specifically a DNA or RNA sequence or a derivative thereof, holdinginformation on that compound or mixture, such as e.g. structure, mass,composition, spatial information etc.

In step 2, all tags of tagged entities are made double stranded by useof a primer (optionally carrying a @-handle such as e.g. biotin),nucleotide triphosphates and a polymerase or transcriptase. Remainingsingle stranded DNA or RNA may optionally be digested by use ofnucleases.

The mixture, is probed for a set of properties in step 3, e.g. but notlimited to affinity for a specific protein. In such a case, entitieswhich do not bind to the specific protein will be eliminated. Anti-tagscomplexed to entities binding to the specific protein may berecovered/be isolated through e.g. the use of its @-handle.

Isolated anti-tags may optionally be amplified in step 4 through the useof PCR or RTPCR.

In step 5, the library of tagged entities of step 1, may undergocomplexation to the isolated and optionally amplified anti-tags of step3 and 4.

Single stranded components are being digested in step 6 by use of e.g.nucleases. The remaining double stranded subset of the library isoptionally subjected to a renewed enrichment of the library according tostep 3-6. Steps 3-6 may be repeated as sufficient number of times toobtain an appropriate chemical entity having the desired property.

In step 7, the isolated anti-tags of step 4 can be cloned and theiridentity be revealed, e.g. in the case of DNA, sequencing may beapplied, whereby the identity of specific entities in the library oftagged entities is revealed.

FIG. 28 relates to a method involving a digestion of single strandedoligonucleotides. In a first step a number of entities (denoted byletters A, B, C . . . ), being it mixtures or single compounds, areattached to a unique tag, holding information on that compound ormixture, such as e.g. structure, mass, composition, spatial informationetc.

In step 2, mixtures of tagged entities are combined with a set ofcomplementary anti-tags. Anti-tags may be, but is not limited tonucleotide derivatives. Anti-tags may optionally carry a @-handle. Thetag and the anti-tags are allowed to form a complex. The complexationmay be, but is not limited to hybridization. Some anti-tags will notform a complex with a tagged entity and some tagged entities will notform a complex with an anti-tag.

Non-complexed components is digested in step 3 using e.g. nucleases whenthe tags and/or anti-tags are composed of DNA or RNA or hybrids thereof.

The mixture of step 3, is probed for a set of properties in step 4, e.g.but not limited to affinity for a specific protein. In such a case,entities which do not bind to the specific protein will be eliminated.Anti-tags complexed to entities binding to the specific protein may berecovered/be isolated through e.g. the use of its @handle. Step 4 may berepeated one or more times.

Isolated anti-tags may optionally be amplified through the use of PCR orRTPCR as illustrated in step 5. Anti-tags may then also be used asdescribed in FIGS. 24-27.

The isolated anti-tags may be cloned and their identity be revealed instep 6, e.g. in the case of DNA, sequencing may be applied, whereby theidentity of specific entities in the library of tagged entities will berevealed.

According to FIG. 29, step 1, a number of entities (denoted by lettersA, B, C . . . ), being it mixtures or single compounds, are attached toa unique tag more specifically a DNA or RNA sequence or a derivativethereof, holding information on that compound or mixture, such as e.g.structure, mass, composition, spatial information etc.

All tags of tagged entities are made double stranded in step 2 by use ofa primer (optionally carrying a @-handle such as e.g. biotin),nucleotide triphosphates and a polymerase or transcriptase. Remainingsingle stranded DNA or RNA may optionally be digested by use of e.g.nucleases.

In step 3, the mixture is probed for a set of properties, e.g. but notlimited to affinity for a specific protein. In such a case, entitieswhich do not bind to the specific protein will be eliminated. Anti-tagscomplexed to tags having appended entities binding to the specificprotein may be recovered/be isolated through e.g. the use of its@-handle. Step 3 may be repeated one or more times.

According to step 4, isolated anti-tags may optionally be amplifiedthrough the use of PCR or RTPCR. Anti-tags may then also be used asdescribed in FIGS. 24-27.

The isolated anti-tags may be cloned in step 5 and their identity berevealed, e.g. in the case of DNA, sequencing may be applied. Whereby,the identity of specific entities in the library of tagged entities willbe revealed.

FIG. 30, step 1, produces a number of entities (denoted by letters A, B,C . . . ), being it mixtures or single compounds which are attached to aunique tag more specifically a DNA or RNA sequence or a derivativethereof, holding information on that compound or mixture, such as e.g.structure, mass, composition, spatial information etc.

In step 2, the mixture is probed for a set of properties, e.g. but notlimited to affinity for a specific protein. In such a case, entitieswhich do not bind to the specific protein will be eliminated. Step 2 maybe repeated.

All tags of tagged entities are made double stranded in step 3 by use ofa primer (optionally carrying a @-handle such as e.g. biotin),nucleotide triphosphates and a polymerase or transcriptase. Remainingsingle stranded DNA or RNA may optionally be digested by use of e.g.nucleases.

Anti-tags complexed to tags of entities binding to the specific proteinmay be recovered/be isolated in step 4 through e.g. the use of its@-handle. Anti-tags may optionally be amplified through the use of PCRor RTPCR. Anti-tags may then also be used as described in FIGS. 24-27.

The isolated anti-tags may be cloned in step 5 and their identity berevealed, e.g. in the case of DNA, sequencing may be applied, whereby,the identity of specific entities in the library of tagged entities isrevealed.

FIG. 31A shows the result of polyacrylamide gel electrophoresis of thesample created in example 13, the result of annealing identifier oligoE57 with zipper building block E32 and anti-codon oligonucleotideCD-M-8-01720001 (with anti-codon sequence Anti-Codon 1) (lane 2) and ofannealing the same identifier oligo with E32 and anti-codon oligo E60(with anti-codon sequence Anti-codon X) (lane 3).

FIG. 31B shows the result of polyacrylamide gel electrophoresis of thesample in which E58 is annealed to zipper building block CX-1 andanti-codon oligo CD-M-8-0172-0001, and E58 to E32 and E60. This time areactant on the zipper building block was cross linked to the displaymolecule in the identifier oligonucleotide.

FIGS. 32 and 33 are more fully discussed in Example 14.

FIGS. 34-47 are more fully discussed in examples 1-5, 7-9.

EXAMPLES Example 1 Loading of a Scaffold onto Identifier Molecules

An amino-modifier C6 5′-labeled identifier oligo(5′-X-TCGTAACGACTGAATGACGT-3′, (SEQ ID NO: 5) wherein X may be obtainedfrom Glen research, cat. #10-1039-90) was loaded with a peptide scaffold(Cys-Phe-Phe-Lys-Lys-Lys, CFFKKK, SEQ ID NO: 6) using SPDP activation(see below). The SPDP-activation of amino-oligo was performed using 160μl of 10 nmol oligo in 100 mM Hepes-KOH, pH=7.5, and 40 μl 20 mM SPDPand incubation for 2 h at 30° C. The activated amino-oligo was extracted3 times with 500 μl EtOAc, dried for 10 min in a speed-vac and purifiedusing micro bio-spin column equilibrated with 100 mM Hepes-KOH. Theloading of scaffold was then performed by adding 10 μl of 100 mMattachment entity and incubating overnight at 30° C.

The loaded identifier oligo was precipitated with 2 M NH₄OAc and 2volume 96% ethanol for 15 min at 80° C. and then centrifuged for 15 minat 4° C. and 15.000 g. The pellet was re-suspended in water and theprecipitation was repeated. Wash of the oligo-pellet was done by adding100 μl of 70% ethanol and then briefly centrifuged. The oligo wasre-dissolved in 50 μl H₂O and analysed by MS. The MS analysis wasperformed after 100 pmol oligo in 10 μl water was treated with 10 μl ofion exchanger resin and incubated minimum 2 h at 25° C. on a shaker.After incubation the resin was removed by centrifugation and 15 μl ofthe supernatant was mixed with 7 μl of water, 2 μl of piperidine andimidazole (each 625 mM) and 24 μl acetonitrile. The sample was analysedusing a mass spectroscopy instrument (Bruker Daltonics, Esquire3000plus). The observed mass, as can be seen in FIG. 34, was 7244.93 Da,which correspond well with the calculated mass, 7244.00 Da. Thisexperimental data exemplify the possibility to load scaffolds ontoidentifier oligonucleotides. This loaded identifier molecule can be usedto receive functional entities from building blocks. This particularscaffold harbours three identical reactive groups, i.e. the amine groupof the lycin side chain, and can therefore be transferred with one, two,or three functional entities, which is capable of reacting with theamine groups.

In the above figure, the DNA sequence is SEQ ID NO:5 and the peptidesequence (which, in the figure, is in reverse order, C-terminal toN-terminal is SEQ ID NO:6.

Example 2 Loading of Functional Entities onto Building Blocks

Loading of functional entities onto building block molecules can be doneusing a thiol-oligo (see below). An Biotin 5′ labeled and thio-modifierC6 S—S (obtainable from Glen Research, cat #10-1936-90) 3′-labeledbuilding block oligo (5′-BTGCAGACGTCATTCAGTCGTTACGA-3′ SEQ ID NO: 7) wasconverted to an NHS-oligo using NHM.

10 nmol oligo was dried in speed-vac, re-dissolved in 50 μl 100 mM DTT,100 mM sodium-phosphate pH 8.0 and incubated at 37° C. for 1 hour. Thethiol-oligo was then purified using micro bio-spin column equilibratedwith 100 mM Hepes-KOH, pH 7.5. The thiol-oligo was converted toNHS-oligo by adding 100 mM NHM in 100 mM Hepes-KOH pH. 7.5. The samplewas incubated at 25° C. over night. The NHS-oligo was then purifiedusing bio-spin column equilibrated with MS-grade H₂O.

In the above figure, the DNA sequence is SEQ ID NO:7.

The MS analysis was performed after 100 pmol oligo in 10 μl water wastreated with 10 μl of ion exchanger resin and incubated minimum 2 h at25° C. on a shaker. After incubation the resin was removed bycentrifugation and 15 μl of the supernatant was mixed with 7 μl ofwater, 2 μl of piperidine and imidazole (each 625 mM) and 24 μlacetonitrile. The sample was analysed using a mass spectroscopyinstrument (Bruker Daltonics, Esquire 3000plus). The observed mass ascan be seen in FIG. 35 was 8369.32, which correspond well with thecalculated mass, 8372.1. The experimental data exemplify the possibilityto convert the attachment entity on building block oligonucleotides.This product can later be used to attach transferable functionalentities.

The NHS-oligo was then used to load functional entities. EDC activationof the functional entity (4-pentynoic acid) was performed mixing 50 μlof 200 mM functional entity in DMF with 50 μl of 200 mM EDC in DMF andincubated for 30 min at 25° C. on a shaker. The loading was thenperformed using 1 nmol NHS-oligo lyophilized in a speed-vac and 10 μl ofthe activated building block (see below). This was incubated at 25° C.for 5 min and then mixed with 30 μl 100 mM MES pH. 6.0. The loadedNHS-oligo was purified using bio-spin column equilibrated with 100 mMMES pH 6.0. The loaded building block oligo is then used immediately forthe transfer reaction without any MS analysis. This is due to theunstable structure of the functional entity during the conditions usedfor the MS measurements.

In the above Figure, the DNA sequence is SEQ ID NO:7.

This experiment exemplifies a complete loading of a functional entityonto a building block molecule ready for transfer to an recipientreactive group when annealed to the complementary identifier molecule.

Another example of a functional entity that can be loaded as describedabove onto a building block is a 5-hexynoic acid as shown below. Again,no MS analysis was performed on this compound due to the unstablestructure of the functional entity in the conditions used in the MSmeasurements.

In the above figure, the DNA sequence is SEQ ID NO:7

Example 3 Transfer of Functional Entities from the Building Block to theIdentifier Molecule

The attachment entity (AE) in the following experiments are either ascaffold, e.g. the peptide, CFFKKK (SEQ ID NO: 134), loaded on anidentifier as prepared in Example 1 or a recipient reactive groupexemplified by an amino modified oligonucleotide used as startingmaterial in Example 1. These attachment entities allow transfer of threeor one functional entities, respectively.

The identifier used in this experiment is an identifier oligonucleotideloaded with CFFKKK as described in Example 1. The functional entity (FE)in this experiment is the 4-Pentynoic acid, the loading of which wasdescribed in Example 2. The identifier molecule loaded with the scaffoldis annealed to the loaded building block molecule to bring theattachment entity and the functional entity in close proximity. Theannealing is directed by the identifier region in the identifiermolecule and the complementary sequence in the building block molecule.

(SEQ ID NO: 5 AE-TCGTAACGACTGAATGACGT       + (SEQ ID NO: 7)FE-AGCATTGCTGACTTACTGCAGACGTB       ↓ (SEQ ID NO: 5)AE-TCGTAACGACTGAATGACGT (SEQ ID NO: 7) FE-AGCATTGCTGACTTACTGCAGACGTB

After the annealing step between the identifier and building blockmolecules, the transfer reaction takes place where the functional entityis transferred to the identifier molecule.

The annealing was performed using 600 pmol of the building block and 400pmol identifier molecules in 0.1 M MES buffer at 25° C. in a shaker for2 hours. The reactive part (functional entity) of the building block wastransferred to the one of the amino group on the attachment entity onthe identifier molecule during the annealing (see below). Afterannealing the sample was purified by micro-spin gel filtration andanalyzed by MS. The sample was prepared for MS analysis using equalamount of sample (about 100 pmol) and ion exchanger resin and incubatedminimum 2 h at 25° in a shaker. After incubation the resin wascentrifuged down and 15 μl of the supernatant was added 7 μl of water, 2μl of piperidine and imidazole (each 625 mM) and 24 ul acetonitrile. Thesample was analysed on a Mass Spectroscopy instrument (Bruker Daltonics,Esquire 3000plus). The observed mass (see FIG. 36) was 7323.45 Da, whichcorrespond well with the calculated mass, 7324.00 Da. Thus, the MSspectrum of the identifier molecule after the transfer reaction shows amass corresponding to the transferred functional entity on theidentifier molecule.

In the above figure, the first sequence is SEQ ID NO:5 and the second isSEQ ID NO:7.

Another example of transfer of functional entity is shown below usingthe amino oligo directly as the AE on the identifier molecule. Thefunctional entity on the building block molecule used in this experimentwas 4-pentynoic acid, as disclosed in example 2.

The annealing was performed using 500 pmol of the building block and theidentifier molecules in 0.1 M MES buffer and incubating the mixture at25° C. in a shaker for 2 hours. The reactive part (functional entity) ofthe building block was transfer to the amino group on the identifiermolecule during the annealing (see below). After annealing and transferthe sample was purified by micro-spin gel filtration and analyzed by MS.The sample was prepared for MS analysis using equal amount of sample(about 100 pmol) and ion exchanger resin and incubated minimum 2 h at25° in a shaker. After incubation the resin was removed bycentrifugation and 15 μl of the supernatant was added 7 μl of water, 2μl of piperidine and imidazole (each 625 mM) and 24 ul acetonitrile.

In the above figure, in both the starting materials and the products,the first sequence is SEQ ID NO:5 and the second is SEQ ID NO:7.

The sample was analysed on a Mass Spectroscopy instrument (BrukerDaltonics, Esquire 3000plus). The observed mass was 6398.04 Da, whichcorrespond well with the calculated mass, 6400.00 Da. Thus, the MSspectra of the identifier molecule after transfer of the functionalentity show a mass corresponding to the transferred functional entity onthe identifier molecule. This example shows that functional entities canbe transferred using this setup of a building block molecule and anidentifier molecule.

Another example of transfer of functional entity is shown below usingthe amino oligo directly as the identifier molecule. The functionalentity used in this experiment was 5-Hexynoic acid, prepared as shown inexample 2.

The annealing was performed using 500 pmol of the building block and 500pmol of the identifier molecules in 0.1 M MES buffer incubated at 25° C.in a shaker for 2 hours. The reactive part (functional entity) of thebuilding block was transferred to the amino group on the identifiermolecule (see below). After annealing and transfer the sample waspurified by micro-spin gel filtration and analyzed by MS. The sample wasprepared for MS analysis using equal amount of sample (about 100 pmol)and ion exchanger resin and incubated minimum 2 h at 25° C. in a shaker.After incubation the resin was removed by centrifugion and 15 μl of thesupernatant was added 7 μl of water, 2 μl of piperidine and imidazole(each 625 mM) and 24 ul acetonitrile.

In the above figure, in both the starting materials and the products,the first sequence is SEQ ID NO:5 and the second is SEQ ID NO:7.

The sample was analysed on a Mass Spectroscopy instrument (BrukerDaltonics, Esquire 3000plus). The observed mass was 6411.96 Da, whichcorrespond well with the calculated mass, 6414 Da. Thus, the MS spectraof the identifier molecule after transfer of the functional entity showa mass corresponding to the transferred functional entity onto theidentifier molecule. This example shows that functional entities can betransferred using this setup of a building block molecule and anidentifier molecule.

Example 4 Extension of the Identifier Molecule to Transfer Unique Codons

After the transfer of the functional entity (FE) to the attachmententity (AE) on the identifier molecule, the identifier molecule isextended in order to transfer the unique codon, that identifies thetransferred functional entity, to the identifier molecule. This isaccomplished by adding a suitable polymerase and a polymerase buffercontaining the wild type nucleotides (dATP, dTTP, dCTP, dGTP). This willextend the identifier molecule in the 3′-end towards the end of the5′-end of the building block molecule. The extension of the identifiermolecule to transfer the unique anticodon(s) is preferably performedafter the transfer of the FE as shown below.

(SEQ ID NO: 5) FE-AE-TCGTAACGACTGAATGACGT (SEQ ID NO: 7)  —AGCATTGCTGACTTACTGCAGACGTB               ↓ (SEQ ID NO: 166)FE-AE-TCGTAACGACTGAATGACGTCTGCT (SEQ ID NO: 7)  —AGCATTGCTGACTTACTGCAGACGTB

The extension was performed using 15 units Taq polymerase in a buffercontaining 0.4 mM of each nucleotide in an extension buffer (20 mMHEPES-KOH, 40 mM KCl, 8 mM MgCl₂, pH=7.4). After the extension reactionthe sample was analyzed using MS. The MS analysis was performed usingabout 100 pmol purified extension mixture in a half volume of ionexchanger resin and incubated minimum 2 h at 25° C. in a shaker. Afterincubation the resin was removed by centrifugation and 15 μl of thesupernatant was mixed with 7 μl of water, 2 μl of piperidine andimidazole (each 625 mM) and 24 μl acetonitrile. The sample was analysedon a Mass Spectroscopy instrument (Bruker Daltonics, Esquire 3000plus).

The MS data for extension on the identifier molecule with a transferred4-Pentynoic acid is shown FIG. 39.

The observed mass was 7922.53 Da, which correspond well with thecalculated mass, 7924.00 Da. The MS spectra of the identifier moleculeafter the transfer reaction of the functional entity and extensionreaction of the encoding region (the unique codon) showed a masscorresponding to the transferred functional entity and the extension onthe identifier molecule. This example shows that functional entities canbe transferred using this setup with a longer building block moleculethan the identifier molecule and that the identifier molecule can beextended using a polymerase after the transfer process. This shows thepossibility to transfer both the functional entity and the unique codonfrom the same building block to an identifier molecule.

Another example showing transfer and extension is for the building blockwith the functional entity 5-Hexynoic acid. The MS data for extension onthe identifier molecule with a transferred 5-Hexynoic acid is shown inFIG. 40.

The observed mass was 7936.99 Da, which correspond well with thecalculated mass, 7938.00 Da. The MS spectra of the identifier moleculeafter transfer reaction of the functional entity and extension reactionof the encoding region (the unique codon) showed a mass corresponding tothe transferred functional entity and the extension on the identifiermolecule. This example also shows that functional entities can betransferred using this setup with a longer building block molecule thanthe identifier molecule and the identifier molecule can be extendedusing a polymerase after the transfer process. This exemplifies thepossibility to transfer both the functional entity and the unique codonfrom one building block molecule to one identifier molecule.

Example 5 Library Design

The identifier molecule can be designed to operate optimal under variousconditions. However, it should contain a few elements that are vital forthe function. The identifier molecule should comprise of a sequence thatcan anneal to the building block and an attachment entity that canaccommodate various functional entities. Below is an example on how anidentifier molecule can be designed in the extension region. The regionthat becomes extended during each step of transfer and encoding can bedesigned using various approaches. Importantly, there must be abase-pair match between the building block and the identifier to allowefficient extension using a polymerase. This can be accomplished usingeither a region that is constant, the binding region as described inFIG. 3 (A), or a region that allow binding to any given sequence, alsoshown in FIG. 3 (B). A combination of these to approaches can also beused.

The first step in the extension process needs no special binding regiondue to the match of the identifier and the building block molecules(step 1 shown below). However, the subsequently steps needs a bindingregion sufficient complementary to the identifier molecule to allow forhybridisation because the enzyme, preferably a polymerase must be ableto bind to the duplex and perform an extension. The example below showsfour steps in the encoding procedure. This process of extension can becontinued to obtain the suitable number of transfer of building blocks.The binding region in this example contains 6 nucleotides, but this canbe varied dependent on the design of the building blocks.

A possibility to accommodate the possible mismatches in the previousanticodon is to use universal nucleobases, i.e. a nucleobases with theability to base pair with more than one of the natural nucleobases. Apossible base is inosine which can form base pairs with cytidine,thymidine, and adenosine (although the inosine:adenosine pairingpresumably does not fit quite correctly in double stranded DNA, so theremay be an energetic penalty to pay when the helix bulges out at thispurine:purine pairing). In principle, any design that allows extensionof the unique codons is possible to use

1.-161. (canceled)
 162. A split-and-mix method for generating a libraryof bifunctional complexes comprising a display molecule part and acoding part, which method comprises the steps of: providing in separatecompartments nascent bifunctional complexes, each comprising a chemicalreaction site and a priming site for enzymatic addition of anoligonucleotide tag; performing in any order reaction in eachcompartment between the chemical reaction site and one or morereactants, and addition of one or more respective oligonucleotide tagsidentifying the one or more reactants at the priming site using one ormore enzymes; pooling together the content of two or more compartmentsand subsequently splitting the pooled contents into an array ofcompartments for a new round of reaction; performing a new round ofreaction, the end product of a preceding round of reaction being used asthe nascent bifunctional complex, to obtain a library of bifunctionalcomplexes in which each member of the library comprises a reagentspecific reaction product and respective oligonucleotide tags which codefor the identity of each of the reactants that have participated in theformation of the reaction product.
 163. The method of claim 162, whereinoligonucleotide tags are double stranded during a reaction of thechemical reaction site with one or more reactants.
 164. The method ofclaim 162, wherein two or more oligonucleotide tags consist of a DNAbackbone structure.
 165. The method of claim 164, whereinoligonucleotide tags consisting of a DNA backbone structure are added tothe priming site by a DNA ligase.
 166. The method of claim 165, whereinligation of oligonucleotide tags is performed in a double strandedstate.
 167. The method of claim 166, wherein the enzyme is a T4 DNAligase or a Taq DNA ligase.
 168. The method of claim 162, wherein thecoding part of the bifunctional complexes consist of double strandedDNA.
 169. The method of claim 162, wherein the coding part comprisingall the oligonucleotide tags is transformed to a double stranded form byan extension method in which a primer is annealed to the 3′ end of anoligonucleotide and extended using a polymerase.
 170. The method ofclaim 169, wherein the extension method is performed by a combination ofa ligase and a polymerase.
 171. The method of claim 162, wherein thelibrary contains from 10³ to 10⁶ bifunctional complexes.
 172. The methodof claim 162, wherein the library contains from 10³ to 10¹⁰ bifunctionalcomplexes.
 173. The method of claim 172 comprising the further step ofpartitioning the library of different bifunctional complexes, saidmethod comprising the step of targeting a target entity and selectingfrom the library of bifunctional complexes those complexes which have anaffinity for said target.
 174. The method of claim 173, wherein theselection is repeated one or more additional times.
 175. The method ofclaim 173, wherein the target is immobilized.
 176. The method of claim173, wherein the partitioning step involves size exclusionchromatography.
 177. The method of claim 173 comprising the further stepof amplifying the oligonucleotide identifier of the selectedbifunctional complexes.
 178. The method of claim 173 comprising thefurther step of sequencing the oligonucleotide identifier.
 179. Themethod of claim 172 comprising the further step of partitioning thelibrary of different bifunctional complexes, said method comprising thestep of targeting a target entity and selecting from the library ofbifunctional complexes those complexes which have an affinity for saidtarget.